Methods and compositions for genetically modifying primate bone marrow cells

ABSTRACT

A method is provided for genetically modifying primate bone marrow cells, comprising isolating bone marrow cells from a primate and, by means which enhance the local concentration of retroviral particles, contacting the isolated bone marrow cells to cells that produce a recombinant amphotropic retrovirus with a genome based on a retroviral vector that contains the genetic information to be introduced into the bone marrow cells. Recombinant amphotropic retrovirus-producing cells, suitable for use in this method also are provided, as are genetically modified primate bone marrow cells with the capacity for regeneration in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No. 08/820,479, filed Mar. 18, 1997, pending, now U.S. Pat. No. ______, which is a continuation-in-part of application Ser. No. 08/211,342, filed Mar. 24, 1994, now U.S. Pat. No. 5,612,206 issued Mar. 18, 1997, the contents of which is incorporated herein by reference.

TECHNICAL FIELD

[0002] The invention concerns the field of gene therapy and more particularly relates to a method for genetically modifying primate bone marrow cells so that they have the capacity to regenerate in vivo, and to cells that produce recombinant retroviral vectors that can be used in such a method. The method is exemplified by the use of means which enhance the local concentration of retroviral particles derived from the murine leukemia virus in the vicinity of target primate stem cells.

BACKGROUND

[0003] Developments in the field of molecular biology have led to a better understanding of the genetic basis underlying the development of a large number of disorders. It is expected that the genes which are associated with the diseases that occur most frequently will have been identified, cloned and characterized before the end of this century.

[0004] So far, molecular genetics has contributed to medicine by the development of diagnostic tools and methods and the biotechnological production of pharmaceuticals. It may be expected, however, that it will also be possible to use the increasing knowledge of genetics for an essentially new therapeutic treatment, the so-called gene therapy. The purpose of gene therapy is to treat disorders by genetically modifying somatic cells of patients. The uses of gene therapy are not limited to hereditary disorders; the treatment of acquired diseases is also considered to be one of the possibilities. Although this field of study is still in a preliminary stage and must be developed, therapeutic possibilities are in the distance which can drastically improve medicine in the future (Anderson, Science 226:410 (1984); Belmont and Caskey, in Gene Transfer, R. Kucherlapati, eds, Plenum press, New York and London, p. 411 (1986); and Williamson, Nature 298:416 (1982)).

[0005] An important cell type for gene therapy purposes is the so-called hematopoietic stem cell which is the precursor cell of all circulating blood cells. This stem cell can multiply itself without losing its differentiating ability. In adult animals most stem cells are situated in the bone marrow. Very infrequently, stem cells also start to circulate in the peripheral blood. This phenomenon can be significantly augmented by treatment with stem cell mobilizing agents, including but not restricted to certain hematopoietic growth factors. In embryos, stem cells are by nature circulating much more frequently. Thus, bone marrow, peripheral blood after stem cell mobilization and embryonic blood, for example, collected perinatally from the umbilical vein, are useful sources for stem cells. The underlying idea of a gene therapy directed to hematopoietic stem cells is that gene transfer to (a limited number of) stem cells may already be sufficient to replace the entire blood-forming tissue with genetically modified cells for a lifetime (Williamson, supra). This would enable treatment not only of diseases that are caused by a (hereditary) defect of blood cells, but also of diseases that are based on the inability to make a certain protein: the modified blood (forming) system could be a constant source of the protein, which could do its work at the places where necessary. It is also possible, with the introduction of genetic material into the blood system, to obtain resistance against infectious agents, to combat cancer, or even to overcome a predisposition to chronic diseases, such as rheumatism or diabetes. Finally, it is noted that in the treatment of some diseases it is to be preferred or necessary that the gene transfer to stem cells is performed on bone marrow cell populations from which certain cell types have been removed. One could for instance consider the use of gene therapy in the treatment of leukemia, in which case there should not occur any gene transfer to the leukemic cells.

[0006] One of the conditions for providing of such a bone marrow gene therapy protocol is a technique by which genes can be incorporated into the chromosomes of target cells, in such a manner that those genes are also passed on to the daughter cells and that the desired protein product is produced in those cells. In the invention described herein, for this purpose use is made of recombinant retroviruses that carry with them the genes to be introduced and which are capable of delivering them to mammalian cells. They make use of the natural characteristic of retroviruses to integrate efficiently and stably into the genome of the infected cell, but not themselves to cause a productive infection because the retroviruses used are replication-defective and are not contaminated with wild-type viruses (Temin, Gene Transfer, supra).

[0007] The recombinant retroviruses which are used within the framework of the present invention are derived from viruses with a natural host-specificity that includes primates, or from viruses that can be pseudotyped with a host-specificity that includes primates. The viruses include, but are not restricted to, murine leukemia viruses (MuLV; Weiss et al., RNA Tumor Viruses, New York (1984)) with a so-called amphotropic or xenotropic host-range, gibbon ape leukemia viruses (GaLV; Lieber et al., Proc. Natl. Acad. Sci. USA 72, 2315-2319 (1975)), and primate lentiviruses.

[0008] For the production of recombinant retroviruses, two elements are required: the so-called retroviral vector, which, in addition to the gene (or genes) to be introduced, contains all DNA elements of a retrovirus that are necessary for packaging the viral genome and the integration into the host genome; and the so-called packaging cell line which produces the viral proteins that are necessary for building up an infectious recombinant retrovirus (Miller, Hum. Gene Ther., 1:5 (1990)).

[0009] As the presence of replication-competent viruses in a gene therapy protocol is considered highly undesirable, most modem packaging cell lines are constructed in a way such that the risk of recombination events whereby a replication-competent virus is generated, is minimized. This is effected by physically separating into two parts the parts of the virus genome that code for viral proteins and introducing them into the cell line separately (Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460 (1988); Markowitz et al., J. Virol. 62:1120 (1988); and Markowitz et al., Virology 167:400 (1988)).

[0010] As the presence of both constructs is essential to the functioning of the packaging cell line and chromosomal instability occurs regularly, it is of great importance for the routine use of such cells in gene therapy procedures that, by means of a selection medium, selection for the presence of the constructs can be provided for. Therefore, these constructs are often introduced by means of a so-called cotransfection whereby both viral constructs are transfected together with a dominant selection marker. The possibility of selection thus provided is certainly not a trivial requirement, considering for instance the observation that we and various other research groups made, that virus-producing cells based on the packaging cell line ψCRIP (Danos & Mulligan, Proc. Natl. Acad. Sci. USA 85:6460 (1988)) are not stable. That is to say that they are no longer resistant to the relevant selection media and during cultivation lose their capacity to produce retroviruses. One example, of importance for one of the embodiments of the present invention, is the so-called POC-1 cell line which was produced by us on the basis of ψCRIP cells (Van Beusechem et al., J. Exp. Med. 172:729 (1990)) which on account of its instability cannot be used for gene therapy on a routine basis. Therefore, in the invention described here, use is made of packaging cells which, by means of a dominant selection culture, do continue to produce stable virus.

[0011] Studies in mice have demonstrated that using amphotropic retroviral vectors, bone marrow stem cells can be provided with a new gene. After transplantation of these modified cells into lethally irradiated mice, the new gene could also be demonstrated for long periods in many different blood cell types of the transplanted animals (Van Beusechem et al., supra).

[0012] Previous problems with regard to the nonexpression of the newly introduced genes were solved by us by using a retroviral vector in which the expression of the gene of choice, is driven by a retroviral promoter whose expression-specificity has been changed by means of a replacement of the so-called enhancer (Van Beusechem et al., supra; and Valerio et al., Gene 84:419 (1989)). In the present invention, these vectors are called LgXL (ΔMo+PyF101), wherein X represents the code of a gene yet to be filled in.

[0013] Before the results obtained from research into gene transfer into the blood-forming organ of mice can be translated into an eventual use of gene therapy in the clinic, a number of essential questions must be answered by studying a relevant preclinical model. First of all, it has to be demonstrated that efficient gene transfer is also possible to blood-forming stem cells of higher mammals, in particular primates. Moreover, genetic modification coupled with autologous bone marrow transplantation in primates requires complex logistics which cannot be studied in mice. The organization of the blood-forming organs of mice and humans can only be compared to a certain extent and it will be clear that the sizes of the two species, and hence the numbers of cells involved in transplantation, differ considerably.

[0014] The experimental animal model is eminently suitable for preclinical gene therapy studies is the non-human primate, in particular the rhesus monkey, partly because the current bone marrow transplantation protocols in the clinic are principally based on data obtained from experiments with bone marrow from the rhesus monkey. Gene therapy procedures using bone marrow cells can be tested in this animal model by taking bone marrow, modifying this genetically by means of recombinant retroviruses and subsequently transplanting it back autologously (i.e., into the same monkey) after the endogenous bone marrow cells have been eradicated by irradiation.

[0015] To date, such experiments have met with little success with regard to:

[0016] a) the hematopoietic regeneration that could be effected with the infected bone marrow, and

[0017] b) the in vivo stability of the genetic modification.

[0018] In the studies published to date, gene transfer was performed either by incubating bone marrow cells with cell-free virus supernatant harvested from virus-producing cells or by a direct exposure of the bone marrow cells to the virus-producing cells. The latter method involves a so-called co-cultivation wherein the virus-producing cells are adhered to the bottom of a culture bottle and the bone marrow cells are seeded on top thereof. Following co-cultivation, the non-adherent bone marrow cells are subsequently harvested and used as transplants.

[0019] In the first reported study (Anderson et al., “Gene transfer and expression in non-human primates using retroviral vectors,” in Cold Spring Harbor Symposia on Quantitative Biology, Volume LI, eds. Cold Spring Harbor Laboratory, New York, p. 1073 (1986); and Kantoff, P. W., A. P. Gillio, J. R. McLachlin, C. Bordignon, et al., J. Exp. Med. 166:219 (1987)), in 19 monkeys an autologous transplantation was performed with bone marrow cells infected with different retroviral vectors containing the gene for neomycin resistance (neo^(r)) or dihydrofolate reductase (DHFR), or with a virus in which neo^(r) and the gene for adenosine deaminase (ADA) are located together, produced by cells that also produce replication-competent virus. Both gene transfer methods described above, i.e., the co-cultivation procedure and the infection with virus supernatant that can be harvested from the virus-producing cells were utilized. Using the co-cultivation procedure, it was not possible to obtain hematopoietic regeneration after autologous transplantation. As a result, only three out of the 13 monkeys survived this procedure. None of the surviving monkeys showed any signs of genetic modification in vivo. Complete hematopoietic reconstitution could be obtained in the six monkeys that received supernatant-infected bone marrow and in four of these animals the gene could be demonstrated. However, genetic modification remained low and transient. Nor could it be precluded that the observed modification had occurred in long-living T-cells which did not generate from the bone marrow cultured in vitro, but were already present as a contaminant in the infected bone marrow.

[0020] In the second study (Bodine et al., Proc. Natl. Acad. Sci. USA 87:3738 (1990)), bone marrow from rhesus monkeys was co-cultivated with cell lines that produce neo^(r)-containing viruses. In this study, also, only the provirus could be demonstrated in vivo after infection by means of a virus-producing cell line that produces contaminatory helper viruses. In this setting, no long-term studies could be performed because again the bone marrow proved incapable of reconstituting the hematopoietic system.

[0021] In conclusion, in the data published so far, the co-cultivation method has always been associated with a drastic loss of in vivo regenerating capacity of the bone marrow cells (Anderson et al., “Gene transfer and expression in non-human primates using retroviral vectors,” in Cold Spring Harbor Symposia on Quantitative Biology, Volume LI, eds. Cold Spring Harbor Laboratory, New York, p. 1073 (1986); Kantoff, P. W., A. P. Gillio, J. R. McLachlin, C. Bordignon, et al., J. Exp. Med. 166:219 (1987); and Bodine et al., supra), so that a clinical application is precluded.

[0022] In addition, none of the studies published to date are sufficiently interpretable as regards genetic modification, since they invariably involved the use of virus preparations in which replication-competent virus was present. Via a so-called “rescue,” this may lead to a spread of the recombinant virus genome after the cells have been transplanted, so that it remains unclear whether the modified cells are offspring of infected bone marrow cells. The present invention provides a method for efficient gene transfer into primate hematopoietic stem cells without a significant loss of the in vivo regenerating capacity of the isolated cells.

[0023] Relevant Literature

[0024] Fibronectin as a single molecule has been reported to bind retroviruses and hemopoietic cells, thereby enhancing the gene transfer efficiency (WO 95/26200).

BRIEF SUMMARY OF THE INVENTION

[0025] The invention provides a method for genetically modifying primate hematopoietic stem cells. The method includes the step of combining isolated primate hematopoietic stem cells with a recombinant retrovirus using means which increase the local concentration of recombinant retrovirus particles in the vicinity of the stem cells over that which is obtained in the absence of such means, so that the chance of infection of the stem cells is enhanced. The recombinant retrovirus contains genetic information to be introduced into the hematopoietic stem cells and has a host range which includes primate hematopoietic stem cells. In some cases, it is preferred that the isolated hematopoietic cell population is enriched for hematopoietic stem cells before the hematopoietic stem cells are brought in close physical contact with the recombinant retrovirus. It is preferred that the genome of the recombinant retrovirus is based on a retroviral vector which is derived from a viral MuLV vector. It is furthermore preferred that the recombinant retrovirus has an amphotropic host range. According to the invention, the “close physical contact” provides an efficient genetic modification of the primate hematopoietic stem cells. The close physical contact can be accomplished by various means, which are exemplified in the different embodiments of the invention. Those skilled in the art will be able to use other means to achieve close physical contact without departing from the present invention.

[0026] The term “hematopoietic stem cell” is understood to mean a cell that has the following characteristics: (1) it has the ability to differentiate into any type of cell of the blood cell system, and (2) it has the capacity to multiply itself without losing its characteristics 1 and 2. The term “hematopoietic cell” is understood to mean any cell of the blood cell system, independent of its lineage commitment or maturation state. Thus, “hematopoietic cells” include “hematopoietic stem cells.” The term “primates” is understood to mean all primates, including man. Preferably, the gene therapy concerns man. By “close physical contact” is intended a contact which enhances the local concentration of retrovirus particles in the direct vicinity of the target cell beyond that obtained under standard conditions, where “standard conditions” are those where retrovirus particles and target cells are mixed together in a liquid solution at normal gravitation.

[0027] In one embodiment of the invention, isolated hematopoietic cells from a primate are, by means of a co-cultivation, exposed to cells that produce the recombinant retrovirus. During this co-cultivation, isolated hematopoietic cells are in the direct vicinity of virus-producing cells. In particular, the hematopoietic stem cells from primates are subject to close contact and these cells adhere, in part or possibly preferentially, to virus-producing cells. During the co-cultivation, virus-producing cells continuously produce new recombinant retroviruses that are shed from the cell membrane into the culture medium. After their production, recombinant retroviruses have a limited life span that depends at least in part on their nature, on the culture temperature and on the composition of the culture medium. Hence, the shorter the distance recombinant retroviruses have to travel from the site where they were shed into the medium towards the isolated hematopoietic cells from a primate the higher is the chance for a successful genetic modification of isolated hematopoietic cells of a primate. In this aspect of the invention, therefore, the most efficient genetic modification is obtained for the subset of isolated hematopoietic cells from a primate that most intimately adhere to virus-producing cells. For this reason, it is preferred that following co-cultivation both nonadherent and adherent cells are harvested.

[0028] In another embodiment, the intimate interaction between virus-producing cells and hematopoietic stem cells is further improved by forcing these cells together. This can be accomplished by various physical means, including but not restricted to increasing the gravitational force to enhance sedimentation of the hematopoietic stem cells onto the virus-producing cells by centrifugation, centrifuging a mixture of both cell populations onto a solid material, concentrating the mixture on the same physical site by electrodiffusion, forcing by pressure or centrifugation the mixture onto a porous solid material with pores large enough in size to allow passage of the fluid medium but small enough in size to prevent passage of the mixture. In the latter application of the invention, the pressure is either positive pressure applied to the fluid medium or negative pressure applied to the porous solid material or to a space past the porous solid material. Alternatively, intimate interaction can also be improved by performing the culture in the presence of a compound that binds both the virus-producing cells and the hematopoietic stem cells.

[0029] In yet another embodiment, hematopoietic stem cells are cultured in recombinant retrovirus containing medium in the presence of a compound that binds both the recombinant retrovirus and the hematopoietic stem cell, thus providing the close physical contact between hematopoietic stem cell and recombinant retrovirus. The compound is characterized by its capacity to bind (1) the hematopoietic stem cell, and (2) the recombinant retrovirus and/or the virus-producing cell. It is preferred that the compound besides binding to the recombinant retrovirus or virus-producing cell preferentially, or even exclusively, binds to the hematopoietic stem cell. In this way, the compound selectively increases the genetic modification of the hematopoietic stem cell. The compound comprises one or more molecules that are selected from or are derived from synthetic or naturally occurring molecules including but not restricted to polymers, antibodies, peptides, cell surface membranes or fragments or components thereof, extracellular matrices or components thereof, intact cells, and complete tissues or components thereof. The molecules include composite molecules containing parts from molecules of different origin. Preferred compounds in the invention are derived from or are components of the natural hematopoietic microenvironment present in the bone marrow of animals. The hematopoietic stem cells by nature closely interact with cells and extracellular matrix molecules present in the hematopoietic microenvironment. In addition, the cells produce cytokines that support the maintenance and functioning of the hematopoietic stem cells and the extracellular matrix molecules bind cytokines that support the maintenance and functioning of the hematopoietic stem cells.

[0030] The method of the invention is also performed using a different kind of compound that (1) binds to the recombinant retrovirus vector and (2) is immobilized on a solid support material. The hematopoietic cells of a primate are brought in close contact with the solid support material (and thereby with the bound recombinant retrovirus vector) by any other means exemplified in the various embodiments of the invention (like gravity, electrodiffusion, and fluid flow).

[0031] In another embodiment of the invention the close contact between the recombinant retrovirus and the hematopoietic stem cell is accomplished by forcing the recombinant retrovirus towards the hematopoietic stem cell by any of various physical means. These include but are not restricted to increasing the gravitational force by centrifugation to induce settling of the recombinant retrovirus onto the hematopoietic stem cell, causing the recombinant retrovirus to move towards the hematopoietic stem cell by electrodiffusion, and forcing the recombinant retrovirus-containing medium through a bed of hematopoietic cells including the hematopoietic stem cell. In the latter case, the hematopoietic cells are seeded on top of a porous solid material with pores large enough in size to allow passage of the fluid medium but small enough to prevent the cells to pass. In this application of the invention, force is provided by either normal gravity, increased gravity through centrifugation, positive pressure applied to the medium, or negative pressure applied to the porous solid material or to a space past the porous solid material. In this aspect of the invention it is preferred but not essential that the solid material used binds the recombinant retrovirus.

[0032] As is clear from the foregoing, the invention provides means to bring isolated hematopoietic cells including hematopoietic stem cells from a primate in close physical contact with a recombinant retrovirus. This is accomplished either directly, by bringing the recombinant retrovirus itself in close proximity of the isolated hematopoietic cells, or indirectly, by bringing cells that produce the recombinant retrovirus in close proximity of the isolated hematopoietic cells.

[0033] According to the invention, it is preferred that the retroviral vector comprises two LTRs long terminal repeats) derived from a viral MuLV vector and the 5′ part of the gag gene of a MuLV. The MuLV sequences are preferably derived from the viral Mo-MuLV vector (Moloney Murine Leukemia Virus), while at least the 3′-LTR is a hybrid LTR which contains the PyF101 enhancer instead of the Mo-MuLV enhancer. To this end, preferably the retroviral vector pLgXL(ΔMo+PyF101) is used, wherein X represents the genetic information to be introduced into the bone marrow cells.

[0034] According to the invention, producer cells that can be used include all recombinant retroviral vector-producing cell lines with a host range that includes primates. Several examples of producer cell lines that produce retroviral vectors with the LgXL(ΔMo+PyF101) structure useful in the invention have been disclosed in PCT International Patent Application WO96/35798. The cells that produce the recombinant retrovirus are preferably recombinant mammalian cells which contain and express the gag, pol and env genes of MuLV. The env gene is preferably derived from an amphotropic MuLV. The gag, pol and env genes of MuLV in the recombinant mammalian cells are preferably distributed over at least two different eukaryotic expression vectors. Further, it is preferred that each packaging construct is associated with a selectable marker gene. As recombinant mammalian cells, GP+envAM 12 cells are preferred, it is further preferred that the cells that produce a recombinant retrovirus contain several copies of the retroviral vector.

[0035] According to the invention, it is further preferred that the cultivation of hematopoietic stem cells in recombinant retrovirus supernatant or with cells that produce recombinant retrovirus occurs in the presence of serum and at least one hematopoietic growth factor. In some embodiments of the invention, it is further preferred to culture the hematopoietic stem cells for a period of time in the absence of recombinant retrovirus and virus-producing cells before being subjected to genetic modification with recombinant retrovirus or to culture the hematopoietic stem cells for a period of time in the absence of recombinant retrovirus and virus-producing cells after having been subjected to genetic modification with recombinant retrovirus.

[0036] The invention further provide cells that produce a recombinant amphotropic retrovirus with a genome based on a retroviral vector, preferably one which is derived from a viral MuLV vector, which contains genetic information that is suitable to be introduced into bone marrow cells of a primate according to the method described herein.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention provides a method for introducing a gene X into isolated hematopoietic cells including hematopoietic stem cells from a primate, whereby the isolated hematopoietic cells are brought in close contact with a recombinant retrovirus. Preferably, the recombinant retrovirus is an amphotropic retrovirus whose genome is composed of the recombinant retroviral vector pLgXL(ΔMo+PyF101) wherein gene X represents a nucleic acid molecule inserted therein that encodes a ribonucleic acid molecule or a protein which is of importance for gene therapy. The invention is comprised of a number of useful components: a recombinant retroviral vector pLgXL(ΔMo+PyF101), a virus-producing cell line shedding recombinant pLgXL(AMo+PyF101) retrovirus, and a method by which isolated hematopoietic cells or purified hematopoietic stem cells of a primate are provided with gene X.

[0038] Hematopoietic Cells

[0039] Many different standard procedures are known in the art for the collection, storage, processing, and reinfusion of hemopoietic cells from bone marrow, peripheral blood, fetal liver, or umbilical cord blood of primates, as well as for conditioning of the recipient and for post-transplantation supportive care (see, e.g., Bone Marrow and Stem Cell Processing: A Manual of Current Techniques, eds. E. M. Areman, H. J. Deeg, and R. A. Sacher, F. A. Davis Company, Philadelphia, pp. 487 (1992); Marrow Transplantation: Practical and Technical Aspects of Stem Cell Reconstitution, eds. R. A. Sacher and J. P. AuBuchon, American Association of Blood Banks, Bethesda, Md., pp. 187 (1992)). Several methods for stem cell enrichment by CD34+cell selection are known in the art that use commercially available materials. They have been compared by Wynter et al., Stem Cells (1995) 13: 524-532. The MACS Cell Sorting method (Miltenyi Biotec, Germany) gives the best results with respect to purity and recovery, and is thus preferred.

[0040] True in vitro tests for hemopoietic stem cells do not exist, but phenotypic analysis is usually performed as an indicator of the quality of both the isolated material and the graft after gene transfer (Knaän-Shanzer et al., Gene Therapy, 3:323-333 (1996)). In the experiments with rhesus monkeys described in the examples this was not done, because not all essential antibodies for this analysis react with rhesus monkey cells. There is no special treatment of the cells prior to cultivation with retrovirus particles.

[0041] Recombinant Retroviral Vector pLgXL(ΔMo+PyF101)

[0042] The recombinant retroviral vector includes DNA elements originating from a MuLV which are necessary in cis for the packaging, reverse transcription and integration of the retroviral genome; these include two so-called Long Terminal Repeats (LTR) and the so-called packaging sequences. In the LTR a modification has been provided by replacing the enhancer originating from MuLV with the enhancer of the polyoma virus strain PyF101 (Linney et al., Nature 308:470 (1984)). In the plasmid construct, it is not necessary that this modification is present in both LTRs; only the 3′ LTR must be provided with the modification since that portion of the LTR ends up in both LTRs after a viral infection (Van Beusechemetal., J. Exp. Med. 172:729 (1990); and Valerio et al., Gene 84:419 (1989)), and the 5′ part of the MuLV gag-encoding sequences such as present in the vector N2 (Armentano, D., S. F. Yu, P. W. Kantoff, T. Von Ruden, W. F. Anderson and E. Gilboa, “Effect of internal viral sequences on the utility of retroviral vectors,” J. Virol. 61:1647 (1987)), so as to effect a higher viral titer. Optionally, the ATG initiation codon of gag can be mutated by means of site-directed mutagenesis, so that it is no longer a translation start site. The only absolute requirements for the vector are (i) the inclusion of DNA elements necessary in cis for the packaging, reverse transcription, and integration of the retroviral genome, and (ii) that the gene X be placed within a proper transcription unit, wherein it is preferred that this transcription unit is a natural viral transcription unit (no internal promoter). The pLgXL(ΔMo+PyF101) vector meets these requirements and includes some further improvements, as exemplified above.

[0043] The retroviral vector is included in a plasmid construct having plasmid sequences necessary for propagation of the vector in E. coli bacteria such as for instance pBR322 (Bolivar et al, Gene 2:95 (1977)) or a vector from the pUC series (Vieira and Messing, Gene, 19:259(1977)); on these, both an origin of replication and a selectable gene (for instance for ampicillin to be of tetracycline resistance) are present, together with gene X. The term “gene” is understood to mean a nucleic acid molecule encoding a ribonucleic acid molecule or protein. It includes naturally occurring nucleic acid molecules and synthetic derivatives thereof. Useful genes that encode a ribonucleic acid molecule or a protein which is of importance for gene therapy include, but are not restricted to, all genes associated with hereditary disorders wherein a therapeutic effect can be achieved by introducing an intact version of the gene into somatic cells. Most of them are documented in: McKusick, Mendelian Inheritance in Man, Catalogs of Autosomal Dominant Autosomal Recessive, and X-Linked Phenotypes, 8^(th) edition, John Hopkins University Press (1988); and Stanbury et al., The Metabolic Basis of Inherited Disease, 5th edition, McGraw-Hill (1983).

[0044] Examples of gene X include: genes associated with diseases of the carbohydrate metabolism such as for: fructose-1-phosphate aldolase; fructose-1,6-diphosphatase; glucose-6-phosphatase; lysosomal α-1,4-glucosidase; amylo-1,6-glucosidase; amylo-(1,4:1,6)-transglucosidase; muscular phosphorylase; liver phosphorylase; muscular phosphofructokinase; phosphorylase-b-kinase; galactose-1-phosphate uridyl transferase; galactokinase; all enzymes of the pyruvate dehydrogenase complex; pyruvate carboxylase; 2-oxoglutarate glyoxylate carboligase; and D-glycerate dehydrogenase; genes associated with diseases of the amino acid metabolism such as for: phenylalanine hydroxylase; dihydrobiopterin synthetase; tyrosine aminotransferase; tyrosinase; histidase; fumarylacetoacetase; glutathione synthetase; γ-glutamylcysteine synthetase; orinithine-δ-aminotransferase; carbamoylphosphate synthetase; ornithine carbamyltransferase; argininosuccinate synthetase; argininosuccinate lyase; arginase; L-lysine dehydrogenase; L-lysine ketoglutarate reductase; valine transaminase; leucine isoleucine transaminase; “branched chain” 2-keto acid decarboxylase; isovaleryl CoA dehydrogenase; acyl-CoA dehydrogenase; 3-hydroxy-3-methylglutaryl CoA lyase; acetoacetyl CoA 3-ketothiolase; propionyl CoA carboxylase; methylmalonyl CoA mutase; ATP:cobalamine adenosyltransferase; dihydrofolate reductase; methylene tetrahydrofolate reductase; cystathionine β-synthase; sarcosine dehydrogenase complex; proteins belonging to the glycine cleavage system; β-alanine transaminase; serum carnosinase; and cerebral homocarnosinase; genes associated with diseases of fat and fatty acid metabolism such as for: lipoprotein lipase; apolipoprotein C-II; apolipoprotein E; other apolipoproteins; lecithin cholesterol acyltransferase; LDL receptor; liver sterol hydroxylase; and “Phytanic acid” α-hydroxylase; genes associated with lysosomal defects such as for: lysosomal α-L-iduronidase; lysosomal iduronate sulfatase; lysosomal heparin N-sulfatase; lysosomal-acetyl-α-D-sulfatase; lysosomal acetyl CoA:α-glucosaminide N-acetyltransferase; lysosomal N-acetyl-α-D-glucosaminide 6-sulphatase; lysosomal galactosamine 6-sulphate sulfatase; lysosomal β-galactosidase; lysosomal arylsulfatase B; lysosomal β-glucuronidase; N-acetylglucosaminylphosphotransferase; lysosomal α-D-mannosidase; lysosomal α-neuraminidase; lysosomal aspartylglycosaminidase; lysosomal α-L-fucosidase; lysosomal acid lipase; lysosomal acid ceramidase; lysosomal sphingomyelinase; lysosomal glucocerebrosidase; lysosomal galactosylceramidase; lysosomal arylsulfatase A; α-galactosidase A; lysosomal acid β-galactosidase; and α-chain of the lysosomal hexosaminidase A; genes associated with diseases of the steroid metabolism such as for: 21-hydroxylase; 11β-hydroxylase; androgen receptor; steroid 5α-reductase; steroid sulfatase; genes associated with diseases of the purine and pyrimidine metabolism such as for: phosphoribosylpyrophosphate synthetase; hypoxanthine guanine phosphoribosyltransferase; adenine phosphoribosyltransferase; adenosine deaminase; purine nucleoside phosphorylase; AMP deaminase; xanthine oxidase; orotate phosphoribosyltransferase; orotidine 5′-phosphate decarboxylase; and DNA repair enzymes; genes associated with diseases of the porphyrin and heme metabolism such as for: uroporphyrinogene III cosynthase; ferrochelatase; porphobilinogene deaminase; coproporphyrinogene oxidase; proporphyrinogene oxidase; uroporphyrinogene ill synthase; uroporphyrinogene decarboxylase; bilirubin UDP-glucuronyltransferase; and catalase; genes associated with diseases of the connective tissue, muscles and bone such as for: lysyl hydroxylase; procollagen peptidase; α1-antitrypsin; dystrophin; alkaline phosphatase; and guano sine nucleotide regulatory protein of the adenyl cyclase complex; genes associated with diseases of the blood and blood-forming organs such as for: blood coagulation factor V; blood coagulation factor VII; blood coagulation factor VII; blood coagulation factor IX; blood coagulation factor X; blood coagulation factor XII; blood coagulation factor XIII; all other blood coagulation factors; all genes associated with osteopetrosis such as for: “carbonic anhydrase II”; thrombocytes membrane glycoprotein lb; thrombocytes membrane glycoprotein IIb-IIIa; spectrin; pyruvate kinase; glucose-6-phosphate dehydrogenase; NADH cytochrome b₅reductase; β-globin; and α-globin; genes associated with diseases of transport systems such as for: lactase; sucrase-α-dextrinase; 25-hydroxyvitamin D₃-1-hydroxylase; and cystic fibrosis transport regulator; genes associated with congenital immunodeficiencies such as for: the proteins of the complement system including B, Clq, Clr, C2, C3, C4, C5, C7, C8 and C10; the inhibitor of Cl, a component of the complement system; the inactivator of C3b, a component of the complement system; genes for X-linked immunodeficiencies such as for: one of the enzymes of the NADPH oxidase complex; myeloperoxidase; and the syndrome of Wiscott Aldrich and Ataxia Telangiectasia; genes coding for hormones as well as the genes coding for their receptors such as for instance for growth hormone.

[0045] Gene X also includes genes which (to date) have not been associated with a hereditary defect but with which gene therapy can be practiced in some manner. These include: the gene for tyrosine hydroxylase, drug resistance genes such as for instance: the P-glycoprotein P170 (the so-called multidrug resistance gene mdr 1); mdr 3; dihydrofolate reductase (DHFR) and methotrexate-resistant isotypes thereof; metallothionine; aldehyde dehydrogenase (ALDH); and glutathione transferase; genes coding for all cytokines including, for instance, all interleukins and all interferons; genes coding for all growth factors; genes coding for all growth factor receptors; genes coding for all transplantation antigens such as, for instance, the major and minor histocompatibility antigens; genes capable of affording resistance against infectious organisms, such as, for instance, TAR decoys (Sullenger et al., Cell 63:601 (1990)), antisense ribonucleic acid molecules, ribozymes, and intracellular antibodies; genes of infectious organisms which can be used for vaccination purposes such as, for instance, the envelope gene of HIV; and genes which can be used for negative selection such as for instance the thymidine kinase gene of the Herpes simplex virus against which selection can be effected with substrates such as, for instance, gancyclovir or acyclovir (Borelli et al., Proc. Natl. Acad. Sci. USA 85:7572 (1988); and Mansour et al., Nature 336:348 (1988)).

[0046] The Virus-Producing Cells

[0047] In order to obtain a stable, selectable virus-producing cell line which produces the amphotropic recombinant retrovirus, pLgXL(ΔMo+PyF101) is introduced into an amphotropic packaging cell line that is selected for the presence of the DNA sequences which are of importance for the production of the viral proteins. One example of such a cell line is GP+envAml2 (Markowitz et al., Virology, 167:400 (1988)). It has been demonstrated, on the other hand, that ψCRIP is not selectable and is unstable with respect to virus production (Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460 (1988)).

[0048] The selectable packaging cell line is based on mammalian cells and produces all viral proteins that are coded by the gag, pol and env genes of MuLV. The env gene must originate from a virus with a tropism including primates, and is preferably derived from an amphotropic MuLV. In order to obtain expression of the aforementioned viral genes, they, while cloned in a eukaryotic expression vector, must be under control of a promoter active in the host, preferably a RNA polymerase II promoter, and be followed by a polyadenylation signal. On these so-called packaging constructs, all three viral genes may be present simultaneously as for instance described by Miller (Miller and Buttimore, Mol. Cell. Biol. 6:2895 (1986)), but the genes may also occur separately on two expression vectors as described by Markowitz (Markowitz et al., Virology 167:400 (1988)). This last is to be preferred because it reduces the chances of recombination events leading to helper virus formation.

[0049] As stated, a useful characteristic of the packaging cell line to be used for this invention is the possibility it provides means of selecting for the presence of the above-mentioned packaging constructs. This can be achieved by effecting a physical association of the packaging constructs with a selectable marker gene. This association can be achieved by combining them in one vector (as done with pGag-Po1GPT in reference (Markowitz et al., J. Virol. 62:1120 (1988)) or by means of a so-called cotransfection (review in for instance (Pellicer et al., Science 209:1414 (1980)). The successfully transfected cells can then be isolated by selecting for the marker gene. Since the cotransfected DNA fragments mostly end up ligated to each other at one place in the genome of the transfected cell (Pellicer et al., Science 209:1414 (1980)), the thus selected cells will mostly contain the packaging construct as well. In view of the fact that ψCRIP cells have been made in this way and, nevertheless, are not selectable, the last procedure is not always successful and the construction of vectors with the marker gene cloned into it is to be preferred.

[0050] As a marker gene, genes coding for a large number of different proteins can be used. Widely used and preferred marker genes are: the neomycin resistance gene (Southern and Berg, J. Mol. Appl. Genet. 1:327 (1982)), the hygromycin resistance gene (Blochlinger and Diggelman, Mol. Cell. Biol. 4:2929(1984)), the E. coli xanthine-guaninephosphoribosyl transferase (gpt) gene (Mulligan and Berg, Science 209:1422 (2980)), the histidinol gene (Hartman and Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)), the herpes simplex virus thymidine kinase gene (Colbére-Garapin et al., Proc. Natl. Acad. Sci. USA 76:3755 (1979)) and the methotrexate-resistant isotype of dihydrofolate reductase (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80:2494(1983)). These genes must also be under control of a suitable promoter, in particular a RNA polymerase II promoter, and be followed by a polyadenylation signal.

[0051] The introduction of pLgXL(ΔMo+PyF101) can be effected by means of various physical techniques such as calcium-phosphate precipitation, electroporation or lipofection (Graham and Van der Eb, Nucl. Acids Res. 15:1311 (1973); Potter et al., Proc. Natl. Acad. Sci. USA 81:7161(1984); Felgner and Ringold, Nature 337:387(1989); Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987)). If the packaging cells cannot be selected for the presence of pLgXL(ΔMo+PyF101), use is made of a selectable marker such as for instance an expression vector of the neomycin resistance gene which is transfected together with pLgXL(ΔMo+PyF101). The successfully transfected cells are then be selected by selecting for the marker gene. Since the DNA fragments mostly end up ligated to each other in one place in the genome of the transfected cell, the thus selected cells will mostly contain the retroviral vector as well.

[0052] A preferred procedure is the introduction of pLgXL(ΔMo+PyF101) via an infection. Since viruses are not capable of infecting packaging cells of the same tropism, use must be made of a version of the recombinant retrovirus with a different tropism which is obtained by introducing the DNA initially via a physical technique into packaging cells with a different tropism. For example, ecotropic virus produced by ecotropic packaging cells transfected with a pLgXL(ΔMo+PYF101) construct can be used to infect amphotropic packaging cells. The infected cells are cloned and then tested for their ability to produce virus.

[0053] Further, it is possible to obtain cell lines producing a higher titer of the virus by introducing several copies of the retroviral vector into the packaging cells using the so-called “ping-pong” method (Bestwick et al., Proc. Natl. Acad. Sci. USA 85:5404 (1988); and Kozak and Kabat, J. Virol. 64:3500 (1990)). In this method, an ecotropic virus-producing cell line is co-cultivated with amphotropic packaging cells, which can give rise to repeated infections. In order to enable the amphotropic cells to be cloned back after this co-cultivation, they must be selectable with selective media in which the ecotropic packaging cells do not survive. By plating the cells in such medium, the proper virus-producing clones can be isolated and subsequently analyzed for their capacity to produce the recombinant virus.

[0054] Method by Which Hematopoietic Cells of a Primate Can Be Provided with Gene X. In Such a Manner That the Regeneration Capacity of the Hematopoietic Cells is Maintained and Autologous Transplantation of the Hematopoietic Cells Gives Rise to a Genetically Modified Hematopoietic System

[0055] The above-mentioned recombinant retroviral vectors are used for the efficient introduction of gene X into hematopoietic cells of primates by bringing the hematopoietic cells in close physical contact with the recombinant retroviral vectors. A key aspect of the invention is the realization that the efficiency of gene transfer is in part dependent on the chance for a recombinant retroviral vector particle to associate with its receptor on the surface of a hernatopoietic target cell. Thus, important factors determining the efficiency of recombinant retroviral vector-mediated gene transfer into hematopoietic cells of primates include (1) the concentration of recombinant retroviral vector particles at the site of the hematopoietic target cell, (2) the density and affinity of receptors for the recombinant retroviral vector on the surface of the target cell, (3) the stability of the recombinant retroviral vector particle, and (4) the stability of the hematopoietic target cell. Information relative to these factors is as follows.

[0056] To optimize the concentration of recombinant retroviral vector particles at the site of the target cell, one can increase the total concentration of recombinant retroviral vector particles in the culture medium, by improving the virus-producing cell line or the virus production and harvest procedure. This invention provides an additional method to increase the concentration of recombinant retroviral vector particles at the site of the hematopoietic target cell, i.e., by establishing a close physical contact between the recombinant retroviral vector and the target cell according to one of several means which are exemplified in detail below.

[0057] For the scope of the invention, the density and affinity of receptors for the recombinant retroviral vector on the surface of a certain target cell are regarded as naturally constant factors. Although perhaps receptor expression or integrity on target cells could be influenced, e.g. by changing the culture conditions, this is not part of the invention. It is realized, however, that receptor densities may intrinsically differ between different cell types significantly. It is furthermore realized that especially for cell types with very few functional receptors for the recombinant retroviral vector, which may include the hematopoietic stem cell, it is important to enhance the chance for a virus-to-cell encounter. This will be even more important when a target cell with few functional receptors is part of a cell mixture containing other cell types having higher functional receptor densities.

[0058] In general, the half-life of infectious recombinant retroviral vector particles under standard culture conditions is low (3-9 hours; e.g., Kotani et al., Hum. Gene Ther. 5, 19-28 (1994); Forestell, et al., Gene Ther. 2, 723-730 (1995)). This half-life can be increased by lowering the culture temperature to 32° C. (Kotani et al., Hum. Gene Ther. 5, 19-28 (1994)). Methods to increase the stability of the recombinant retroviral vector particles are not part of the invention. It is realized, however, that the invention providing an increased chance for a virus-to-cell encounter is of particular importance for retroviral vectors with a short half-life.

[0059] It is of critical importance that a method to transfer a gene X into a certain target cell allows the target cell to retain all of its characteristics. Especially in the case of hematopoietic stem cells of primates this has previously been difficult, if not impossible, to achieve. Gene transfer procedures tested on bone marrow grafts ofprimates led to a dramatic loss of the in vivo regenerating capacity of the grafts (see above). The present invention provides a method for efficient transfer of gene X into hematopoietic stem cells of primates that does not significantly affect the in vivo regenerating capacity of the manipulated graft.

[0060] Numerous different procedures for harvest, processing, storage, shipping, etc. of human hemopoietic cells are in use and known to those of skill in the art. Various means of bringing together of the recombinant retrovirus particles and hematopoietic target cells include the following:

[0061] General Procedure for Recombinant Retroviral-Vector Mediated Gene Transfer into Hematopoietic Cells of Primates:

[0062] The hematopoietic cells of a primate are suspended in a suitable culture medium for hematopoietic cells containing recombinant retrovirus vector particles. The hematopoietic cells are either total mononuclear cells or are cell populations that are enriched for stem cells according to various methods known in the art, including but not restricted to, density separation (Percoll, BSA) and positive selection for CD34+cells (FACS sorting, Dynabeads immunoselection, Miltenyi MACS selection, AIS CELLector flask selection, or CellPro CEPRATE selection) or depletion of cells carrying mature cell type cell surface markers. Many different suitable culture media are commercially available. They include, but are not restricted to DMEM, IMDM, and A-MEM, with 5-30% serum and often further supplemented with, for example, BSA, one or more antibiotics. L-glutamine, 2-mercaptoethanol, hydrocortisone, and hematopoietic growth factors. Recombinant retrovirus vector particles are harvested into this medium by incubating virus-producing cells in this medium. To enhance gene transfer, usually compounds such as polybrene, protamine sulphate, or protamine HCl are added. Usually, the cultures are maintained for 2-4 days and the recombinant retrovirus vector containing medium is refreshed daily. Optionally, the hematopoietic cells are precultured in medium with growth factors but without recombinant retrovirus vector particles for up to 2 days, before adding the recombinant retrovirus vector-containing medium.

[0063] For successful gene transfer it is essential that the target cells undergo replication in culture (without differentiation). Most harvested hemopoietic stem cells are resting cells. Therefore, a stimulus to enter cell cycle during culture is needed. This can be accomplished by adding recombinant hemopoietic growth factors (HGF), including different combinations of HGF such as interleukin-3, interleukin-6, and steel factor (SCF). In cultures with stromal cell support, or other cells which produce the necessary HGF, such as a retrovirus-producing cell line, HGF addition is not needed.

[0064] The method of the invention is performed according to one of the following procedures, either indirectly, by bringing hematopoietic cells of a primate in close physical contact with virus-producing cells, or directly, by bringing hematopoietic cells of a primate in close physical contact with recombinant retroviral vector particles.

[0065] The following are examples of indirect methods which are used.

[0066] i) Co-cultivation of hematopoietic cells of a primate with virus-producing cells. The virus-producing cells and the hematopoietic cells from a primate are mixed at the initiation of the co-culture, or a monolayer of adherent virus-producing cells is established before adding the hematopoietic cells from a primate. The virus-producing cells may have been damaged prior to initiation of the co-culture by, for example, a lethal dose of irradiation, but can be used so long as they continue to shed recombinant retroviral vector particles into the medium. The virus-producing cells have the capacity to bind the hematopoietic cells to their surface. Virus-producing cells based on the commonly used packaging cells derived from mouse fibroblasts have this capacity. Due to the intimate interaction between the virus-producing cell and the hematopoietic cell the recombinant retroviral vectors produced by the virus-producing cell only have to travel a very short distance to reach the hematopoietic cell. It may even occur that a recombinant retroviral vector fuses with the membrane of the hematopoietic cell while being shed from the membrane of the virus-producing cell. The invention thus provides a transduction method that minimizes the time during which the recombinant retroviral vector is exposed to destabilizing components of the environment. The most efficient genetic modification is obtained for the subset of hematopoietic cells of a primate that most intimately adhere to the virus-producing cells. It is preferred that virus-producing cells preferentially, or even exclusively, bind hematopoietic stem cells. In this way, the genetic modification of hematopoietic stem cells is selectively increased. In this embodiment of the invention, it is preferred that the co-cultivation takes place for three to four days in the presence of serum and one or more hematopoietic growth factors such as for instance interleukin 3 (IL-3). Following co-cultivation, both the non-adherent and the adherent cells are harvested from the culture (the last-mentioned cells can be obtained by, for example, trypsinization) and used as the transplant.

[0067] ii) Co-cultivation of hematopoietic cells of a primate with virus-producing cells at increased gravitational force. This embodiment of the invention provides a further improvement of and includes the advantages of the procedure described under (i). Apart from the increased gravitational force, the procedure is performed as described under (i). By increasing the gravitational force two effects are being accomplished, i.e., (1) the intimate interaction between the virus-producing cells and the hematopoietic cells from a primate is further enhanced, and (2) recombinant retrovirus vector particles that have been shed into the culture medium are prevented from traveling away from the hematopoietic cells, thus increasing the local concentration of the particles. The increased gravitational force is achieved by performing the co-cultivation while spinning the container with the culture around an axis of rotation. The axis can intersect the container or be located outside of the container. Useful centrifuges to spin the cultures according to the invention are known in the art. The gravitational force is maximized, but should not exceed the maximal gravitational force that allows functional survival of the virus-producing cells, the recombinant retroviral vector particles and the hematopoietic cells from a primate. Usually, the gravitational force will not exceed 2500 g. As a result of the further increased gene transfer efficiency obtained with this embodiment of the invention, the co-culture duration can be significantly shortened. Usually, this procedure will not be performed for more than eight consecutive hours.

[0068] iii) Co-cultivation of hematopoietic cells of a primate with virus-producing cells with increased inter-cellular contact accomplished by electrodiffusion. This embodiment of the invention provides an alternative improvement of and includes the advantages of the procedure described under (i). Apart from the electrodiffusion, the procedure is performed as described under (i). Because the hematopoietic cells of a primate, the virus-producing cells, and the recombinant retroviral vector particle are all negatively charged they can be forced to move towards a positive electrode. By performing the co-cultivation procedure in an electrophoresis unit, negatively charged cells and vectors are concentrated. This way, two objectives are accomplished, i.e., (1) the intimate interaction between the virus-producing cells and the hematopoietic cells from a primate is further enhanced, and (2) the recombinant retrovirus vector particles that have been shed into the culture medium are prevented from traveling away from the hematopoietic cells, thus increasing the local concentration of the particles. Electrophoresis units useful for this aspect of the invention are known in the art. The electrophoresis unit preferably contains two chambers separated by a semi-permeable membrane, with pore sizes that do not permit passage of cells and vectors. In such a two-chamber electrophoresis unit, co-cultivation is performed in the chamber containing the negative electrode. The voltage applied between the electrodes is maximized, but kept below a value that causes significant damage to the hematopoietic cells of a primate, the virus-producing cells, and the recombinant retroviral vector particles. To further reduce damage, voltage may be applied periodically. Also in this embodiment of the invention, virus-producing cells and hematopoietic cells from a primate are mixed at the initiation of the co-culture, or a monolayer of adherent virus-producing cells is established on the surface of the semi-permeable membrane before adding the hematopoietic cells from a primate.

[0069] iv) Co-cultivation of hematopoietic cells of a primate with virus-producing cells with increased inter-cellular contact accomplished by fluid flow. This embodiment of the invention provides another alternative improvement of an includes the advantages of the procedure described under (i). Apart from the fluid flow, the procedure is performed as described under (i). The fluid flow is brought about by forcing the culture medium through a porous solid material with pores large enough in size to allow passage of the culture medium but small enough in size to prevent passage of the hematopoietic cells of a primate and the virus-producing cells. The pores may or may not allow passage of the recombinant retroviral vectors. The force driving the fluid flow is exercised by normal or increased gravitational force or by positive pressure applied to the culture medium or by negative pressure applied to the porous solid material or to a space past the porous solid material. The increased gravitational force is achieved by performing the co-cultivation while spinning the container with the culture around an axis of rotation. The axis can intersect the container or be located outside of the container. The pressure is established using a pump device. Pump and centrifuge devices useful in this aspect of the invention are known in the art. The rate of the fluid flow depends in part on the size of the pores in the solid material: if the pores allow passage of the recombinant retroviral vectors the rate is at a value that at least compensates for random diffusion of the recombinant retroviral vector; if the pores do not allow passage of the recombinant retroviral vector the rate is maximized; but in no case may the rate exceed the maximal rate that allows functional survival of the virus-producing cells, the recombinant retroviral vector particles and the hematopoietic cells from a primate. Also in this embodiment of the invention, the virus-producing cells and the hematopoietic cells from a primate are mixed at the initiation of the co-culture, or a monolayer ofadherent virus-producing cells is established on the surface of the porous solid material before adding the hematopoietic cells from a primate.

[0070] v) Co-cultivation of hematopoietic cells of a primate with virus-producing cells in the presence of a compound that binds both the hematopoietic cells of a primate and the virus-producing cells. This embodiment of the invention provides another alternative improvement of and includes the advantages of the procedure described under (i). Apart from the compound, the procedure is performed as described under (i). The compound has at least one binding site for the hematopoietic cell of a primate and at least one binding site for the virus-producing cell. The nature of the binding sites may be different or the same. The compound is a soluble molecule or a solid support material or comprises several soluble molecules bound directly or indirectly to each other or comprises one or more soluble molecules bound to the same solid support material. The indirect binding may be via a homogeneous or heterogeneous complex of molecules, via cell surface membranes or fragments or components thereof, via intact cells, or even via a complex mixture of different cells. The mixture of cells may be artificially composed or be derived from naturally occurring cell mixtures or tissues. Thus, it is to be understood that the compound, may, for example, comprise a complete naturally occurring tissue. Another nonlimiting example of a compound in this embodiment of the invention is a tissue culture plastic with a coating that binds to the hematopoietic cells of a primate and the virus-producing cells. In this embodiment of the invention it is preferred that the binding site for a hematopoietic cell of a primate has a binding preference for hematopoietic stem cells over other types of hematopoietic cells.

[0071] Preferred compounds in this aspect of the invention are derived from or are components of the natural hematopoietic microenvironment present in the bone marrow of animals. The hematopoietic stem cells by nature closely interact with cells and extracellular matrix molecules present in the hematopoietic microenvironment. In addition, the cells produce cytokines that support the maintenance and functioning of the hematopoietic stem cells and the extracellular matrix molecules bind cytokines that support the maintenance and functioning of the hematopoietic stem cells. We are using a cultured stroma cell population. This is a complex mixture of cells, extracellular matrix molecules and the cytokines produced by the cultured cells. The stroma culture significantly enhances the recovery of a phenotypically defined candidate human hematopoietic stem cell population (approx. 10-fold). Components of the extracellular matrix include collagens, proteoglycans, fibronectin, laminin, elastin, glycosaminoglycans, thrombospondin, and chondronectin.

[0072] Further preferred compounds in this aspect of the invention comprise parts that are derived from antibodies or from peptides with a defined binding capacity. The peptides may be naturally occurring or artificially synthesized or derived from a combinatorial peptide library, including but not restricted to a library made by phage display. Preferred peptides in this aspect of the invention are derived from proteins that are involved in natural inter-cellular adhesion and/or signal transduction processes, where it is more preferred that the natural processes involve at least one cell type of the hematopoietic system.

[0073] A typical nonlimiting example of a compound according to this aspect of the invention is a tissue culture plate coated with a mixture of antibodies directed against molecules on the surface of the virus-producing cell (e.g., retroviral envelope molecules) and molecules on the surface of the hematopoietic cell (e.g., the CD34 molecule present on the membrane of primitive hematopoietic cells), or with bispecific antibodies directed against both cell populations, or with a mixture of synthetic peptides directed against both cell populations (including, for example, peptides derived from cytokines known to act on the hematopoietic cells by binding to a specific receptor molecule).

[0074] The following are examples of direct methods which are used for bringing hematopoietic primate cells into close physical contact with recombinant retroviral vector particles. These embodiments of the invention make use of cell-free recombinant retroviral vector preparations derived from the culture medium of virus-producing cells that is harvested according to standard procedures known in the art. These procedures may include purification, concentration, and the like.

[0075] vi) Sedimentation of recombinant retrovirus vectors onto hematopoietic cells of a primate at increased gravitational force. The increased gravitational force is achieved by incubating the hematopoietic cells in recombinant retroviral vector containing medium while spinning the container with the culture around an axis of rotation according to the procedure described in embodiment (ii). The gravitational force should at least be higher than the minimal force needed to overcome the random diffusion of the recombinant retroviral vector and should not exceed the maximal gravitational force that allows functional survival of the recombinant retroviral vector and the hematopoietic cells from a primate, where it is preferred that the gravitational force is maximized. Usually, the gravitational force will not exceed 2500 g. The centrifugation time depends on the centrifugation speed and on the height of the column of culture medium above the hematopoietic cells, but typically does not exceed two recombinant retroviral vector half-lives. Optionally, the procedure may be repeated several times with fresh recombinant retroviral vector containing medium.

[0076] vii) Electrodiffusion ofrecombinant retroviral vectors towards hematopoietic cells of a primate. By performing the cultivation of the hematopoietic cells of a primate in recombinant retroviral vector containing medium in an electrophoresis unit the hematopoietic cells of a primate and recombinant retroviral vector particles that are both negatively charged are forced to move in the same direction towards the positive electrode and thus are concentrated. Electrophoresis units useful for this aspect of the invention are known in the art. The electrophoresis unit preferably contains two chambers separated by a semi-permeable membrane, with pore sizes that do not permit passage of the hematopoietic cells of a primate and the recombinant retroviral vectors. In such a two-chamber electrophoresis unit the cultivation is performed in the chamber containing the negative electrode. The voltage applied between the electrodes is maximized, but kept below a value that causes significant damage to the hematopoietic cells of a primate and the recombinant retroviral vector particles. To further reduce the damage, the voltage maybe applied periodically. Also in this embodiment of the invention, the procedure is typically not performed for longer than two recombinant retroviral vector half-lives and may be repeated several times with fresh recombinant retroviral vector containing medium.

[0077] viii) Forcing recombinant retroviral vector particles towards hematopoietic cells of a primate by fluid flow. The fluid flow is brought about by forcing the culture medium containing the recombinant retroviral vectors through a porous solid material with pores large enough in size to allow passage of the culture medium but small enough in size to prevent passage of the hematopoietic cells of a primate. The pores may or may not allow passage of the recombinant retroviral vectors. The force driving the fluid flow is exercised as exemplified above under iv). The rate of the fluid flow may range from the value that compensates for random diffusion of the recombinant retroviral vector to the maximal rate that allows functional survival of the recombinant retroviral vector particles and the hematopoietic cells from a primate. The time during which the fluid flow is maintained typically does not exceed two recombinant retroviral vector half-lives. Optionally, the procedure may be repeated several times with fresh recombinant retroviral vector containing culture medium.

[0078] ix) Culture of hematopoietic cells of a primate in recombinant retroviral vector containing medium in the presence of a compound that binds both the hematopoietic cells of a primate and the recombinant retroviral vector. The compound has at least one binding site for the hematopoietic cell of a primate and at least one binding site for the recombinant retroviral vector. The nature of the binding sites may be different or the same. The compound is selected from or derived from the same molecules and materials characterized above under (v). Also in this embodiment of the invention it is preferred that the binding site for a hematopoietic cell of a primate has a binding preference for hematopoietic stem cells over other types of hematopoietic cells.

[0079] Also in this aspect of the invention, preferred compounds are derived from or are components of the natural hematopoietic microenvironment present in the bone marrow of animals. Further preferred compounds in this aspect of the invention comprise parts that are derived from antibodies or from peptides with a defined binding capacity as characterized above under (v). A typical nonlimiting example of a compound according to this aspect of the invention is a tissue culture plate coated with a mixture of antibodies directed against the envelope molecule on the surface of the recombinant retroviral vector and molecules on the surface of the hematopoietic cell, or with bispecific antibodies directed against the vector and cell, or with a mixture of synthetic peptides directed against the vector (e.g., peptides derived from the receptor for the retrovirus envelope molecule) and the cell.

[0080] x) Binding the recombinant retroviral vector to a compound that is immobilized on a solid support material that is brought in close contact with the hematopoietic cells of a primate by anyone of the means exemplified in embodiments vi-viii or similar procedures. Compounds useful in this aspect of the invention have at least one binding site for the recombinant retroviral vector while being immobilized to the solid support material by any physical or chemical means. The solid support materials include but are not restricted to plastics, silicates, metals, and the like. Additional examples of solid support materials include agarose, sephrose, sephadex, cellulose (acetate), DEAE-cellulose, polyacrylamide, polystyrene, Tosylactivated polystyrene, glass, gelatin, dextran, polyethylene, polyurethane, polyester. A nonlimiting example of this embodiment of the invention is the use of a plastic tissue culture dish (the solid support material) coated by standard procedures known in the art with antibodies directed against the retrovirus envelope protein (the compound). Additional examples of solid support materials, in terms of physical structure: single or multi-layer tissue culture dish or flask, semipermeable membrane, porous or nonporous beads including immunomagnetic beads, and (hollow) semipermeable or nonpermeable fibers. Methods of coating and coupling the compound to the solid support materials include the following.

[0081] For polystyrene a simple adsorption procedure can be followed. Protein dissolved in PBS is incubated for several hours at room temperature with the solid support material. Subsequently, the coated solid support material is washed once or several times in PBS or in PBS with 0.1% (w/v) irrelevant protein such as, for example, albumin.

[0082] For other materials, covalent binding is preferred for efficient coating. Many coupling procedures and useful materials are known in the art and are commercially available, for example, CNBr-activated sepharose (manufactured by Pharmacia) or agarose or dextran can be used to couple ligands containing amino groups by incubating them with ligand dissolved in a bicarbonate or borate buffer at high pH (preferably in the range of 8-10) with a high salt content (preferably approximately 0.5M NaCl) for 2 hours at room temperature or for 10-16 hours at 4° C. Subsequently, excess ligand is washed away with coupling buffer, any remaining active groups are blocked with, for example, 0.1 M Tris-HCl buffer pH 8.0 for 2 hours at room temperature or for 10-16 hours at 4° C., and ionically bound free ligand is washed away by alternatively washing with high and low pH buffer solutions such as, for example, Tris-HCl buffer pH 8.0 with 0.5M NaCl and 0.1M acetate buffer pH 4.0 with 0.5M NaCl. Another example is polystyrene activated by p-toluenesulfonyl chloride treatment (such as the Tosylactivated Dynabeads M-450 manufactured by Dynal). Any protein or glycoprotein can be chemically coupled to this material by incubating the solid support material with the ligand dissolved in a high pH buffer such as 0.5M borate buffer pH 9.5 for 24 hours at room temperature. Unbound ligand is removed by several washes with PBS with 0.1% irrelevant protein (such as albumin). Many alternative coupling procedures and commercially available activated soluble support materials useful in the invention are known in the art (see, for example, Affinity Chromatograph. A Practical Approach, eds. P. D. G. Dean, W. S. Johnson, and F. A. Middle, IRL Press, Oxford, pp. 215 (1985)). Apart from a direct coupling of the ligand to the solid support material, the bond can also be made via a spacer molecule. Many reagents that can be used as spacer molecules have been described. Examples include bis-oxiranes, water soluble carbodiimides, SPDP, and glutaraldehyde.

[0083] Finally, natural intermolecular interactions can be exploited to couple proteins to a solid support material, for example, peptides containing a histidine-tag efficiently interact with materials containing nickel ions.

[0084] The recombinant retroviral vector is bound to the compound by incubating a preparation of the recombinant retroviral vector in the tissue culture dish and the hematopoietic cells of a primate are brought in close contact with the recombinant retrovirus by seeding the hematopoietic cells of a primate in the tissue culture dish, where the contact maybe further enhanced by, for example, increasing the gravitational force.

[0085] Following the transfer procedures, it is not possible to determine the gene transfer into true hematopoietic stem cells in vitro, simply because there is no assay for these cells. However, more mature progenitor cells can be tested in standard colony assays (e.g., McNiece et al., Blood, 72:191-195(1988); Sutherland et al., Blood, 74:1563-1570(1989); Breems et al., Leukemia, 8:1095-1104 (1994)). Furthermore, a candidate stem cell population can be analyzed phenotypically (Knaän-Shanzer et al., Gene Therapy, 3:323-333 (1996)). There are several ways of testing for gene transfer into these cells. When gene X encodes a selectable marker gene, clonogenic assays can be performed in the presence of a selective compound and resistant colonies can be scored to determine expression of the marker gene. If gene X encodes a molecule that can be stained with a fluorescent labeled antibody, or when the product of gene X converts a substrate into a fluorescent product, immunofluorescence or FACS analysis can be performed to demonstrate expression of the transgene. When gene X encodes a transport molecule that pumps a fluorescent substrate in or out of cells, expression of gene X can be measured by FACS analysis. Alternatively, isolated progenitor cell derived colonies or cells sorted on a FACS on the basis of their phenotype can be subjected to PCR analysis specific for the introduced retroviral vector. The latter can be done on any vector irrespective of the nature of gene X.

[0086] Several of the embodiments i-x exemplified above may be combined to further optimize the transfer of gene X into the hematopoietic cells of a primate. It is, therefore, to be understood that any combination of the embodiments is also part of the invention. All modifications within the scope of the invention that may be contemplated by the skilled artisan are also claimed to be part of the present invention. All embodiments of the method of the invention can further be used after the hematopoietic cells of a primate have been enriched for hematopoietic stem cells, which is to be preferred in some cases. Enrichment of hematopoietic cells of primates for hematopoietic stem cells can be accomplished by various methods known in the art.

[0087] Below the invention is illustrated with practical examples. It is to be understood that only certain embodiments of the invention are illustrated and that the examples should not be considered restrictive in character.

EXAMPLES

[0088] Example A describes the production of virus-producing cells and recombinant retroviral vectors useful in the invention. In Example B cells and vectors of Example A are shown to be useful for the introduction of a gene X into hematopoietic cells of primates, without affecting the in vivo regenerating capacity of the graft. Example C1 describes a procedure for the enrichment of isolated hematopoietic cells from a primate for hematopoietic stem cells. In Example C2 the usefulness of the invention for the introduction of a gene X into enriched hematopoietic stem cells of a primate without affecting the in vivo regenerating capacity of the graft is demonstrated. Example D shows efficient transduction of hematopoietic stem cells of a primate by sedimentation of recombinant retroviral vectors onto the hematopoietic stem cells at increased gravitational force. Example E1 shows the production of peptides useful in the invention as recombinant retroviral vector binding compounds, and Example E2 describes how these peptides are used for the transferof gene X into hematopoietic cells of a primate according to the invention. Example F1 discloses a procedure for the establishment of a human stroma cell culture derived from the natural microenvironment present in the human bone marrow, and Example F2 describes the use of this stroma cell culture as a binding compound for the transfer of gene X into hemopoietic cells of a primate.

Example A

[0089] Production of Selectable Stable Recombinant Retrovirus-Producing Cells

[0090] In this practical example, use was made of the retroviral vector construct pLgAL(ΔMo+PyF101) (Van Beusechem et a!., J. Exp. Med. 172:729 (1990)), wherein A represents the human cDNA gene coding for adenosine deaminase (ADA). Twenty micrograms of this construct were transfected to the ecotropic packaging cell line GP+E-86 (Markowitz et al., J. Virol. 62:1120 (1988)), according to the method described by Chen and Okayama (Chen and Okayama, Mol. Cell. Biol. 7:2745 (1987)). Prior to the transfection, the GP+E-86 cells had been cultured in medium containing 15 μg/ml hypoxanthine, 250 μg/ml xanthine and 25 μg/ml mycophenolic acid, so as to select for the preservation of the DNA sequences responsible for the production of the viral proteins. Transfectants that produce a functional human ADA enzyme were isolated by means of a selective culture in medium with a combination of 4 μM xylofuranosyl-adenine (Xyl-A) and 10 nM deoxycoformycin (dCF) (Van Beusechem et al., J. Exp. Med. 172:729 (1990)).

[0091] Then, with the thus obtained cells a ping-pong culture as described by Kozak and Kabat (Kozak and Kabat, J. Virol. 64:3500 (1990)) was initiated. To that end, 5×10³ transfectants were mixed with an equal amount of GP+envAm12 amphotropic packaging cells (Markowitz et al., Virology 167:400 (1988)) and cultured together in α-modified DMEM (Dulbecco's Modified Eagle's Medium) with 10% FCS (Fetal Calf Serum) and 8 μg/ml polybrene. The amphotropic packaging cells were also selected prior to use, for the preservation of the DNA sequences coding for the viral proteins (in the medium as described for GP+E-86 cells, with 200 μg/ml hygromycin B added thereto). The culture was expanded for two weeks, at which time the amphotropic virus-producing cells were recovered using the resistance of these cells against hygromycin B. Individual GP+envAm12 clones that express functional human ADA and produce the viral proteins, were obtained by culturing limited cell numbers in medium containing all the above-mentioned components in the amounts mentioned. In all, 12 of such clones were isolated and tested.

[0092] DNA analysis demonstrated that the clones contained several copies of the retroviral vector. The titer of the virus supernatants produced by the 12 clones was measured by exposing murine fibroblasts to dilutions of these supernatants and subsequently determining the number of fibroblasts that had acquired resistance against Xyl-A/dCF as a result thereof The different clones produced between 3×10³ and 2×10⁵ infective virus particles per milliliter supernatant. The best clones produced 100× more virus than the best amphotropic LgAL(ΔMo+PyF101) virus-producing cell line to date, which had been obtained via a single infection with ecotropic virus.

[0093] In order to obtain some idea about the most promising clone with regard to the use in bone marrow gene therapy procedures, rhesus monkey bone marrow was co-cultivated for three days with each of the 12 virus-producing cell lines. Subsequently, the preservation of the capacity of the bone marrow to form hematopoietic colonies in vitro and the infection efficiency regarding the hematopoietic precursor cells, which are at the origin of these colonies, were determined. With some of the clones, infection efficiencies of up to 40-45% Xyl-A/dCF-resistant precursor cells could be achieved, while none of the clones showed a clear toxicity towards these bone marrow cells.

[0094] On the basis of all aforementioned criteria, a cell line was chosen, which was called POAM-P1. This cell line was used to demonstrate in the practical example described under b the usefulness of the thus obtained virus procedures for the genetic modification of the blood-forming organ of primates.

[0095] Two further constructs based on the pLgXL(ΔMo±PyF101) retroviral vector and including further improvements were used, wherein gene X is the gene encoding human glucocerebrosidase. These vectors were designated IG-GC-2 and IG-GC-4 and their construction is described in detail in patent application WO 96/35798, the contents of which are included herein by reference. IG-GC-2 contains the full length human placental glucocerebrosidase (hGC) cDNA, whereas IG-GC-4 has a 160 nt deletion in the 3′ untranslated region of the hGC cDNA. Recombinant retroviral vector-producing cell lines were generated using the PA317 cell line with amphotropic host range (Miller and Buttimore, Mol. Cell. Biol. 6, 2895-2902 (1986)) and using the PG13 cell line with GaLV host range (Miller et al., J. Virol. 65, 2220-2224(1991)) as described in patent application WO 96/35798. The cell lines were designated PA2 (PA317 with IG-GC-2 construct), PA4 (PA317 with IG-GC-4 construct, PG2 (PG13 with IG-GC-2 construct), and PG4 (PG13 with IG-GC-4 construct).

[0096] To harvest batches of recombinant retroviral vector supernatants, T180 tissue culture flasks were inoculated with 1×10⁶ virus-producing cells in 25 ml DMDM (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and the cells were allowed to grow to 90-100% confluency in 4-5 days at 37° C., 10% CO₂ in a 100% humidified atmosphere. Next, the temperature was changed from 37° C. to 32° C. for a period of 24 hours before the medium was replaced with 50 ml fresh culture medium. After an additional culture period of 48 hours, the virus supernatant was harvested, filtered through a 0.45 μm pore size filter, aliquoted and stored at −80° C. The absence of replication-competent retrovirus (RCR) was tested using a S⁺/L⁻ foci test after amplification on mus dunni cells.

[0097] The recombinant retroviral vector titer issuing from the virus-producing cell lines was established on Gaucher type II fibroblasts (GM 1260). GMO 1260 cells were seeded at a density of 10⁵ cells per 35 mm well (in 6-well plates) in culture medium further supplemented with polybrene (4 μg/ml; Sigma). Twenty-four hours later these cells were infected with 1 ml of recombinant retroviral vector supernatant after which the cells were cultured and expanded for 14 days as above. Next, genomic DNA was isolated as described by Stewart et al. (Cell 38 627-637 (1984)). After digestion with NheI and Southern analysis using a 0.65 kb ³²p-labeled NcoI-BglI hGC fragment according to standard procedures, both an hGC endogenous fragment (19 kb) and a proviral DNA fragment of either 3.4 (IG-GC2) or 3.2 kb (IG-GC4) were visible. Comparison of signal intensities between the proviral DNA fragment and the endogenous DNA fragment by ImageQuant volumetric analysis after phosphor screen autoradiography using a Molecular Dynamics PhosphorImager 400A revealed a ratio of 0.8 (PA2), 0.3 (PA4), 0.4 (PG2), and 0.2 (PG4). Since the hybridization signal of the endogenous band represents 2n DNA, on average 1.6, 0.6, 0.8, and 0.4 provirus copies per cell were present, respectively. Taking into account that the seeded GM01260 cells probably divided once before the virus supernatant was applied, approximate virus titers of 3×10⁵, 1×10⁵, 2×10⁵, and 8×10⁴ were calculated for PA2, PA4, PG2 and PG4, respectively.

Example B Preclinical Test of a Bone Marrow Gene Therapy Procedure in Rhesus Monkeys with the Cell Line Poam-p1 Described in Example A

[0098] Rhesus monkey bone marrow was taken by puncturing the upper legs. The bone marrow so obtained was suspended in HBSS/Hepes with 100 units heparin and 100 μg/ml DNase I. Cells having a density lower than 1.064 g/ml were obtained by successively performing a Ficoll separation and a BSA-density gradient centrifugation (Dicke et al., Transplantation 8:422 (1969)). These operations resulted in an enrichment of the cell population for hematopoietic stem cells by a factor of 10-20. The thus obtained bone marrow cells were introduced, in a concentration of 106 cells per ml, into high glucose (4.5 g/liter) α-modified DMEM, containing 5% heat-inactivated monkey serum, 15 mg/ml BSA (Bovine Serum Albumin), 1.25×10⁻⁵ M Na₂SeO₃, 0.6 mg/ml iron-saturated human transferrin, 1 μg/ml of each of the following nucleosides: adenosine, 2′-deoxyadenosine, guanosine, 2′-deoxyguanosine, cytidine, 2′-deoxycytidine, thymidine and uridine, 1.5×10⁻⁵ M linoleic acid, 1.5×10⁻⁵ M cholesterol, 1×10⁻⁴ M β-mercaptoethanol, 0.4 μg/ml polybrene, 100 μg/ml streptomycin, 100 U/ml penicillin and 50 ng/ml of the recombinant rhesus monkey hematopoietic growth factor IL-3 (Burger et al., Blood 76:2229 (1990)). The thus obtained cell suspension was seeded at a concentration of 2×10⁵ cells per cm² onto a 70-80% confluent monocellular layer of POAM-P1 cells, which had shortly before been exposed to 20 Gray γ-radiation. The bone marrow was co-cultivated with the POAM-P1 cells for 90 h at 37° C. in a moisture-saturated atmosphere of 10% CO₂ in air.

[0099] For the duration of the co-cultivation, the rhesus monkey that had donated the bone marrow was conditioned for the autologous reception of the co-cultivated bone marrow by means of total body irradiation with 10 Gray X-rays, divided over two equal fractions at an interval of 24 h, performed, respectively, 2 days and 1 day prior to the transplantation. On the day of the transplantation, the co-cultivated bone marrow was harvested from the culture, including the bone marrow cells that had adhered to the POAM-P 1 cells or cells that had adhered to the plastic of the culture bottle during cultivation. The latter cells were obtained by means of a trypsinization. A monocellular cell suspension was prepared in a physiological salt solution with 10 μg/ml DNase I and infused into a peripheral vein of the donor monkey.

[0100] In order to determine the in vivo regeneration capacity of the co-cultivated bone marrow, use was made of the semi-quantitative assay described by Gerritsen et al., (Gerritsen et al., Transplantation 45:301 (1988)). This method is based on the observation that the rate at which circulating red and white blood cells regenerate after transplantation of autologous bone marrow cells in lethally irradiated rhesus monkeys depends on the size of the transplant. In particular the kinetics of the appearance of the precursors of red blood cells (reticulocytes) is a good standard in this connection. By determining hematological values in the blood system of the monkeys at regular intervals after the transplantation, it could be established (using the relation described by Gerritsen) that the modified bone marrow had preserved sufficient regenerative capacity and the co-cultivation therefore had no toxic side effects.

[0101] Analysis at the DNA level made it clear that long periods (up to more than a year) after the transplantation, the introduced provirus could be traced in various blood cell types (mononuclear cells and granulocytes). Especially the presence of the introduced gene in the granulocytes is considered ofgreat importance. Since granulocytes, after being generated in the bone marrow, remain in the blood stream only a few hours before being broken down, the presence of the human ADA in these cells demonstrates that a year after transplantation the bone marrow still contains very primitive cells that give rise to the formation of ripe blood cells. Also, functional expression of the introduced human ADA gene in ripe blood cells could be demonstrated. These results constitute clear proof of the fact that through the invention described here stable genetic modification of the hematopoietic system of primates can be obtained.

Example C Preclinical Test of a Bone Marrow Gene Therapy Procedure in Rhesus Monkeys Which Utilizes Purified Hematopoietic Stem Cells Example C1 Enrichment of Primate Bone Marrow CD34+CD1 lb-Stem Cells

[0102] Rhesus monkey bone marrow having a density lower than 1.064 g/ml was obtained as described above under Example B. This cell population was successively depleted for cells carrying the monocytes/granulocytes-marker CD11b and enriched for cells carrying the stem cell/precursor cell-marker CD34. This was performed using immunomagnetic beads, which had been made as follows: first, tosyl-activated polystyrene magnetic beads (Dynabeads 450; Dynal, Oslo) were incubated for 24 h in a 0.5 M borate solution pH 9.5 with 1.25 μg protein A (Pharmacia, Uppsala) per 10⁶ beads. After frequent washing in PBS containing 0.1% BSA, to the beads, now protein A-coupled, saturating concentrations of monoclonal antibodies (anti-CD11b: Mol, Coulter Clone, Hialeah, F1; anti-CD34: ICH3,43) were bound by incubating for 30 min at room temperature. Finally, the beads were frequently washed in HBSS/Hepes and stored at 4° C. until use. The bone marrow cells were incubated for 20 min at 4° C. with 7 anti-CD11b beads per cell in a concentration of 5×10⁷ cells/ml at amaximum. Unbound CD11b-negative cells were stripped from beads and CD 11b-positive cells bound thereto, using a magnetic particle collector (MPC; Dynal) and washed in HBSS/Hepes. The thus obtained cells were incubated for 20 min at 4° C. with 5 anti-CD34 beads per cell again in a concentration of5×10⁷ cells/ml at a maximum. After removal of the CD34-negative cells using the MPC, the bound CD34-positive cells were recovered by means of a competitive elution with an excess of immunoglobulins. To that end, the beads with CD34-positive cells were incubated for 1 h at 37° C. in HBSS/Hepes with 25% bovine plasma (Gibco, Paisley) and 500 U/ml heparin.

Example C2 Introduction of the Construct pLgAL(ΔMo+PyF101) Described under A) into Rhesus Monkey CD34+CD11b-Stem Cells

[0103] The introduction of the human ADA gene into rhesus monkey CD34+CD 11b− stem cells and the autologous transplantation procedure were performed as described under Example b above, the only difference being that the co-cultivation was performed with the previously described cell line POC-1 (Van Beusechem eta!., J. Exp. Med. 172:729 (1990)). As noted, this cell line is unstable and not very suitable for large-scale use. For this present experiment, use could still be made of an early passage which does not have a reduced titer.

[0104] After transplantation all blood cell types regenerated completely, which demonstrates that the gene transfer procedure can also be performed on CD34+CD11b− stem cells without toxic side effects. The presence of the provirus in mononuclear blood cells and in granulocytes could also be demonstrated in these monkeys during the entire experimental period (at this point 266 days and 280 days after transplantation in two monkeys) which is still in progress. Expression of the functional human ADA enzyme could also be demonstrated in blood cells of these monkeys. The enrichment for hematopoietic stem cells prior to the gene transfer did not have any demonstrable effect on the efficiency of the gene transfer to stem cells. This experiment therefore demonstrates that the results as described under b) can also be achieved when the bone marrow has been stripped from most riper cell types, which is preferred in some uses of genetic modification of bone marrow cells.

Example D Introduction of the JG-GC Constructs Described under (A) into Human CD34+ Hematopoietic Stem Cells by Increased Gravitational Force

[0105] Bone marrow cells were obtained by aspiration of the iliac crest of normal healthy donors or of a patient with non-Hodgkin Lymphoma. Mononuclear cells were obtained by Ficoll gradient separation according to standard procedures. CD34+ hematopoietic stem cells were isolated using a magnetic antibody separation system (Mini Macs, Milteny) according to the procedures supplied by the manufacturer. This procedure yielded 60-95% pure CD34+ populations with recoveries ranging from 50-90% of the CD34+ cells present in the total bone marrow aspirate.

[0106] Recombinant retroviral vector supernatant of the virus-producing cell lines PA2, PA4, PG2 and PG4 obtained as described under Example (a) was used for the transduction of the isolated human CD34+ cells. The isolated CD34+ cells were seeded at a cell density of 1×10⁵ cells/cm² in 24-well tissue culture plates (Greiner) in 400 μl virus supernatant supplemented with 50 ng/ml interleukin-3 (Sandoz) and 4 μg/ml protamine sulfate (Novo Nordisk Pharma). The plates were subsequently centrifuged for 2.5 hours at 1100 g at room temperature, either once or four times (once daily after refreshing the virus supernatant). After each centrifugation, the cultures were placed overnight at 37° C., 10% CO₂ in a 100% humidified atmosphere. In a control experiment, the cells were cultured for four days as above without the 2.5 hours centrifugation steps. Instead, the recombinant retroviral vector medium was refreshed daily after a 5 minute centrifugation of the cultures at 200 g. The theoretical multiplicity ofinfection of functional recombinant retroviral vector particles in the total culture medium over hematopoietic target cells at the start of the procedure after each supernatant addition was 1.2, 0.4, 0.8, and 0.3 for PA2, PA4, PG2 and PG4 virus, respectively. As a control virus preparation, culture supernatant of the IGvp010 cell line (see patent application WO 96/35798) was used that contains pLgXL(ΔMo+PyF101) derived recombinant retrovirus vectors carrying the human multi-drug resistance (MDR]) gene at a titer of approximately 105 particles per ml as established by vincristine resistant colony formation of human bone marrow cells.

[0107] In experiment 1, CD34+ cells from bone marrow of a non-Hodgkin lymphoma patient were transduced by four incubations with PA2, PG4, or IGvp010 virus supernatant with or without transduction enhancement by centrifugation. After the transduction procedure, transduced CD34+ cells were seeded in 1 ml DMEM (Gibco BRL) with 10% FBS supplemented with 200 ng/ml SCF, 100 ng/ml IL-6, 100 ng/ml IL-3, 100 U/ml GM-CSF, and 100 ng/ml G-CSF. After a 10 day culture period at 37° C., 10% CO₂ in a 100% humidified atmosphere the expanded cells, representing the mature myeloid progeny of the transduced CD34+ cell population, were washed once with PBS and lysed in buffer containing 50 mM potassium phosphate buffer, pH 6.5, 0.1% Triton X-100. Following sonication and centrifugation at 4° C., the clear supernatant was transferred to new tubes, protein concentrations were measured (DC-Biorad kit) and lysates were stored at −20° C.

[0108] Glucocerebrosidase activitywas determined with either4MU-b-glucoside (Sigma) or PNP-b-glucoside (Sigma) as artificial substrate on 20 mg total protein of transduced cells according to described procedures (Aerts et al., Eur. J. Biochem. 150, 565-574 (1985); Havenga et al., BioTechniques 21, 1004-1007 (1996)). TABLE 1 Comparison of 4x supernatant transduction procedure to 4 x centrifugation enhanced transduction Recombinant Retrovirus Relative hGC Activity PCR-positive CFU-GM Vector Supernatant Supernatant Centrifugation Supernatant Centrifugation IGvp010 (negative control) (1) (1) 0/24 (0%) 0/24 (0%) PA2 hGC vector  1  1 1/24 (4%) 5/24 (21%) PG4 hGC vector  1.3  4.5 3/24 (13%) 3/24 (13%)

[0109] Table 1 shows the relative glucocerebrosidase activity data of this experiment, where the results of cells subjected to transduction with the JGvp010 retrovirus vector were set at a value of 1. As can be seen, an increase in hGC activity could not be detected following transduction with PA2 virus, with or without centrifugation. In contrast, PG4 virus transduction couldbe measured by functional hGC activitywhich was 3.5-fold increased following centrifugation (4.5 versus 1.3 in the control). Successful transduction was further confirmed by performing PCR specific for the IG-GC-2 and IG-GC-4 constructs on CFU-GM clonogenic progenitor cell derived colonies. CFU-GM were obtained by seeding 5000 transduced CD34+ cells in 1 ml of methylcellulose medium with cytokines (Methocult GF H4534; Stemcell Technologies, Inc., Vancouver, Canada) in 6-well plates. After 14 days, individual colonies were picked and DNA was isolated as described (van Beusechem et al., Proc. Natl. Acad. Sci. USA 89, 7640-7644(1992)). PCR analysis was performed on this suspension using oligonucleotide primers 5′-CAGCCCATGTTCTACCAC-3′ (SEQ ID NO: 1) and 5′-GGATCCCTAGGCTTTTGC-3′ (SEQ ID NO:2). A 50 μl PCR reaction typically contained 25 pmol of each oligonucleotide, 3% DMSO, 5 μl 10-times concentrated buffer provided with the enzyme, 20 pmol dNTP, and 0.25 Units SuperTaq (Promega). The cycler program consisted of 5 min. 95° C. predenaturation followed by 26 cycles of each 45 sec. 95° C., 1 min. 54° C., 1 min. 72° C. The program was ended by an elongation step of 10 min. at 72° C. Of the PCR product, 10 μl was run on a 1.5% agarose gel, transferred to a membrane and hybridized with a 0.4 kb ³²p-labeled BamHI hGC fragment according to standard procedures. As can be seen in Table 1, all transductions with hGC retrovirus vectors yielded PCR-positive colonies, whereas transductions with the MDR1 control vector did not.

[0110] In experiment 2, PA2, PA4, and PG2 recombinant retroviral vector supernatants (and IGvpO10 control supernatant) were used to transduce CD34+cells from normal healthy donor bone marrow. The centrifugation procedure was performed either once or four times on subsequent days as above. One day after the transduction procedure, CFU-GM were plated for PCR analysis as above. As can be seen in Table 2, all transductions led to high percentages of hGC-vector containing CFU-GM even after a single 2.5 hour centrifugation step. TABLE 2 Comparison of a single centrifugation enhanced transduction procedure to four repeated centrifugation enhanced transduction procedures PCR-positive CFU-GM/number tested (%) Recombinant Retrovirus Four rounds of Vector Supernatant Single transduction transduction IGvp010 control vector 0/20 (0%)  0/20 (0%) PA2 hGC vector 6/20 (30%)  5/20 (25%) PA4 hGC vector 5/20 (25%)  6/20 (30%) PG2 hGC vector 9/20 (45%) 10/20 (50%)

Example E Efficient Transfer of Gene X into Human Cd34+ Hematopoietic Stem Cells Using Recombinant Retroviral Vectors Bound to a Tissue Culture Dish Example E1 Production of Peptides Derived From Receptors For Retroviruses (GLVR)

[0111] The Gibbon ape Leukemia Virus Receptor (GLVR) proteins are transmembrane molecules expressed on the surface of mammalian cells. Their primary function is import of inorganic phosphate and sodium. To date, two different but homologous receptors have been described by means of expression cloning of complementary DNA copies of their murine and human mRNA counterparts (Johann, et al, J. Virol. 66, 1635-1640 (1992); van Zeijl, et al., Proc. Natl. Acad. Sci. USA 91, 1168-1172 (1994); Weiss and Tailor, Cell 82, 531-533 (1995)). The cDNA predicted amino acid sequences and deduced hydropathy plots suggest that both these GLVR1 and GLVR2 proteins traverse the cellular membrane 10 times and have 5 extracellular loops and 4 intracellular loops. The human GLVR1 receptor confers permissivity to Gibbon ape Leukemia Virus and Feline Leukemia Virus-B, whereas the human GLVR2 or amphotropic virus receptor confers permissivity to amphotropic Murine Leukemia Viruses carrying the 4070A or 10A1 envelope molecules. The GLVR1 homologues from different rodent species have small amino acid differences in their 4th extracellular domain which determine virus susceptibility of a cell. Recombinant chimeras between GLVR1 and GLVR2 proteins suggest that the 4th extracellular domain in GLVR1 is involved in virus binding and infection. Therefore, we have synthesized peptides encompassing sequences from the 4th extracellular domain of GLVR1 and GLVR2 using Fmoc chemistry (performed under contract at Research Genetics, Inc. Huntsville, Ala., USA). The amino acid sequences of the peptides read from N-terminus to C-terminus: LVYDTGDVSSKV (SEQ ID NO:3) and LIYKQGGVTQEA (SEQ ID NO:4) for GLVR1 and GLVR2, respectively.

[0112] For certain applications of the invention, the C-terminus of thepeptides is extended with 6 Histidine-residues. This enables coupling to solid support materials via nickle molecules.

Example E2 The Use of GLVR-Peptides as Recombinant Retrovirus Vector Binding Compounds to Enhance the Transfer of Gene X into Human Hematopoietic Stem Cells

[0113] Non-tissue culture dishes, i.e. culture dishes not treated to enhance cell adherence (35 mm; Greiner) are incubated for 2 hours at room temperature with 2 ml 100 μM GLVR1 or GLVR2 peptide in phosphate buffered saline (PBS). This solution was prepared from a 10 mM stock in DMSO. Next, the dishes are washed once with PBS. Two ml recombinant retrovirus supernatant harvested from the PA2 cell line and from the PG4 cell line as described under (d) is incubated at 4° C. for 2 hours on GLVR2 or GLVR1 peptide coated dishes, respectively. This procedure is repeated twice. Optionally, the thus coated dishes are washed with PBS with 1% (w/v) human serum albumin (PBS/HAS) and stored at −80° C.

[0114] Human CD34+ hematopoietic stem cells are obtained as described under Example D, above, are suspended 1×10⁶ cells/ml in IMDM (Gibco BRL) supplemented with 50 ng/ml interleukin-3 (Sandoz), 5% heat-inactivated autologous human serum, 4 μg/ml protamine sulfate (Novo Nordisk Pharma) and 100 U/ml penicillin (Gist-Brocades) and are cultured for 48 hours at 37° C., 10% CO₂ in a 100% humidified atmosphere in non-tissue culture dishes. Next, the cultured cells are placed in the GLVR peptide and recombinant retroviral vector coated dishes in their original culture medium (2 ml/dish) and cultured for another 24 hours at 37° C., 10% CO₂ in a 100% humidified atmosphere. After this culture, all cells including any adherent cells are harvested, washed once in PBS/HAS, and used for analysis of gene transfer or for transplantation by infusion into a peripheral vein.

Example F Enhanced Transfer of Gene X into Human CD34+Hemopoietic Stem Cells Using Recombinant Retrovirus Vectors in the Presence of Human Bone Marrow Stroma

[0115] Example F1

Establishment of Human Bone Marrow Stroma

[0116] Bone marrow mononuclear cells from healthy donors are obtained as described in Example D. Five x10⁷ cells are seeded in T75 Nunclon culture flasks (Nunc, Roskilde, Denmark) in 10 ml DMEM (Gibco) supplemented with 10% heat-inactivated FCS, and cultured at 37° C., 10% CO₂ in a 100% humidified atmosphere. Twenty-four hours later the entire medium, including all nonadherent cells, is removed and replaced with the same medium further supplemented with 2 mM L-glutamine (Gibco), 10⁻⁴ M P-mercaptoethanol (Merck, Darmstadt, Germany), and 10⁻⁵ M hydrocortisone (Sigma) (“stromamedium”). Once aweek, the stromamedium is replaced with fresh stroma medium. After 3-5 weeks, a confluent monolayer of cells is formed. Thereafter, confluent monolayers are trypsinized with Trypsin-EDTA solution (Gibco) and split 1:10 in stroma medium each time after reaching confluence. Each reseeding step is regarded as one passage and includes 3-4 cell doublings. Three individual stroma lines were established and these have now been cultured for 40, 40, and 65 passages, respectively. The three lines exhibited similar functional properties in supporting maintenance of human hemopoietic stem cells throughout the entire study, i.e., at least during the culture period ranging from passage 5 to passage 30.

Example F2 The Use of Human Bone Marrow Stroma as a Binding Compound to Enhance the Retroviral Vector-Mediated Transfer of Gene X into Human Hemopoietic Stem Cells

[0117] Twenty-four-well tissue culture plates are precoated with 0.3% gelatine (Sigma) in PBS for 16 hours at 4° C. Stroma cells are seeded 2×10⁵ cells/well into these plates. After 1-3 days, confluent stroma monolayers are irradiated with 25 Gy γ-radiation. Immediately thereafter, the irradiated stroma monolayers are used to support retroviral vector-mediated gene transfer.

[0118] Serum-free recombinant retrovirus supernatant is harvested from the cell line IGvp010 (see Patent Application WO 96/35798) that produces pLgXL(ΔMo+PyF101) derived recombinant retroviral vectors carrying the MDR1 gene, either in IGTM (alpha-modified DMEM containing 1.5% BSA (Sigma), 1 μg/ml of each of the nucleosides adenosine, 2′-deoxyadenosine, guanosine, 2′-deoxyguanosine, cytidine, 2′-deoxycytidine, thymidine, and uridine (all Sigma), 1.5×10⁻⁷ M Na₂SeO₃ (Sigma), 0.6 mg/ml iron-saturated transferrin (Behring, Marburg, Germany), 1.5×10⁻⁷ M linoleic acid (Sigma), 1×10⁻⁴ M 2-mercaptoethanol (Merck, Darmstadt, Germany) with 5% fetal calf serum (FCS) or in serum-free StemPro-34 SFM Complete Medium (Gibco RBL Life Technologies, Grand Island, N.Y.). The retroviral vector supernatant is flash-frozen and stored at −80° C. until use.

[0119] Human CD34+ bone marrow cells are obtained as described in Example D. They are suspended 1×10⁶ cells/ml in IGvp010 supematant in IGTM with 5% FCS or StemPro-34 SFM Complete Medium supplemented with 50 ng/ml interleukin-3 (Gist-Brocades, Delft, NL) and 1.6 μg/ml protamine HC1 (Kabi Pharmacia, Woerden, NL) and are seeded 5×10⁵ cells per well onto the irradiated stroma monolayers. Control cultures are started with the same cell suspensions in culture dishes without stroma monolayers. The cells are cultured for four days at 37° C., 10% CO₂ in a 100% humidified atmosphere, and each day the complete medium is replaced by fresh IGvp010 supernatant and supplements. On day 4, all cells are harvested by trypsinization as above, and are used for gene transfer analysis or transplantation.

[0120] For analysis of gene transfer into hemopoietic stem cell and progenitor cell populations, the cells are stained with a phycoerythrin-conjugated anti-CD34 MoAb and with a cocktail of fluorescein isothiocyanate-conjugated Moabs directed against CD38, CD33, and CD71 as described (Knaän-Shanzer et al., Gene Therapy, 3:323-333 (1996)). Populations of CD34^(bright)CD33 38,71^(negative) cells, CD34^(positve)CD33,38,71^(positive) cells, CD34^(negative)CD33,38,71^(positive) cells, and CD³⁴ ^(negative)CD33,38,71^(negative) cells are each sorted separately on a FACStar Plus flow cytometer (Becton Dickinson, Mountain View, Calif.). Aliquots of the sorted samples are used for reanalysis to determine the purity of the sorted cells. The presence of the recombinant retroviral vector genome in the sorted cells is determined by a semi-quantitative PCR assay. To this end, untransduced bone marrow mononuclear cells are added to the sorted cell samples to reach a total of 10⁶ cells per sample. The cells are pelleted by centrifugation and DNA is isolated from these cells as described (van Beusechem et al., Proc. Natl. Acad. Sci. USA, 89:7640-7644 (1992)). The isolated DNA concentration is measured using PicoGreen DNA Quantitation reagent (Molecular Probes, Eugene, Oreg.) and of each isolate five independent titrations are prepared containing DNA equivalents down to 10 cells per sample. All samples are subjected to PCR analysis specific for the human MDR1 cDNA gene. The sequences of the primers used are: 5′-GTCACCATGGATGAGATTGAG-3′ (SEQ ID NO:5) (upstream primer) and 5′-CCACGGACACTCCTACGAG-3′ (SEQ ID NO:6) (downstream primer). The reaction conditions are: 10 mM Tris-HCl pH 9.0, 50 mM KC1, 0.01% (w/v) gelatin, 0.1% Triton X-100, 1.5 mM MgCl₂ with 200 μM of all four dNTPs, 200 μM of both primers, and 0.25U SuperTaq polymerase (HT Biotechnology Ltd. Cambridge, UK) in a total volume of 50 μl. Forty cycles of 1 minute at 94° C., 1 minute at 55° C. and 1 minute at 72° C. are performed in 96-well plates using a Biometra UNO-Thermoblock thermocycler. The reaction products are separated on 0.8% agarose gel, blotted, and subject to Southern analysis with human MDR1 gene-specific probes according to standard procedures (Sambrook et al. Molecular Cloning. A Laboratory Manual. 2^(nd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The frequency of PCR-positive cells is determined on the basis of the detection of specific amplification products in the five independent titrations. The outcome of this analysis is corrected for a possible contribution to the PCR signal by any contaminating cells with a different phenotype using the data from the FACS reanalysis of the sorted samples. (See Knaän-Shanzer et al. Gene Therapy, 3:323-333 (1996).

[0121] Our invention shows in an example that bone marrow cells co-cultivated with the virus-producing cells described here are capable of genetically modifying the hematopoietic system of primates after autologous transplantation. This modification was observed for a prolonged period in several blood cell types including granulocytes, which have a very short life time (approximately 8 hours). With the method described by us, these results can also be obtained when the bone marrow has previously been enriched for hematopoietic stem cells by removal of most other (riper) bone marrow cells. These data demonstrate our capacity to infect very primitive cells and show that it is possible to carry out gene therapy using such modified bone marrow cells.

[0122] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0123] The invention now having been fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

1 6 1 18 DNA Artificial Sequence misc_feature Description of Artificial Sequence primer specific for the IG-GC -2 and IG-GC-4 constructs which contain the full length human placental glucocerebrosidase (hGC) cDNA and the hGC cDNA with 160 nucleotide deletion in the 3′ region, respectively 1 cagcccatgt tctaccac 18 2 18 DNA Artificial Sequence misc_feature Description of Artificial Sequence primer specific for the IG-GC-2 and IG-GC-4 constructs which contain the full length human placental glucocerebrosidase (hGC) cDNA and the hGC cDNA with 160 nucleotide deletion in the 3′ region, respectively 2 ggatccctag gcttttgc 18 3 12 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on the 4th extracellular domain of Gibbon ape Leukemia Virus Receptor protein 1. 3 Leu Val Tyr Asp Thr Gly Asp Val Ser Ser Lys Val 1 5 10 4 12 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on the 4th extracellular domain of Gibbon ape Leukemia Virus Receptor protein 2. 4 Leu Ile Tyr Lys Gln Gly Gly Val Thr Gln Glu Ala 1 5 10 5 21 DNA Artificial Sequence misc_feature Description of Artificial Sequence primer specific for the human multi drug resistance gene 1 (MDR1) cDNA 5 gtcaccatgg atgagattga g 21 6 19 DNA Artificial Sequence misc_feature Description of Artificial Sequence primer specific for the human multi drug resistance gene 1 (MDR1) cDNA 6 ccacggacac tcctacgag 19 

What is claimed is:
 1. A method for genetically modifying primate hematopoietic stem cells, said method comprising: combining isolated primate hematopoietic stem cells which can undergo replication with a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted, wherein said combining uses means whereby said hematopoietic stem cells and said recombinant retrovirus are brought into close physical contact, so that chance of infection of said hematopoietic stem cells by said recombinant retrovirus is significantly enhanced.
 2. The method according to claim 1, wherein said means comprises co-cultivation of said hematopoietic stem cells with cells which produce said recombinant retrovirus.
 3. The method according to claim 1, wherein said means are physical means.
 4. The method according to claim 3, wherein said physical means comprise centrifugation of medium containing said hematopoietic stem cells and said recombinant retrovirus.
 5. The method according to claim 1, wherein said means comprise use of a compound which has binding affinity for both said recombinant retrovirus and said hematopoietic stem cells.
 6. The method according to claim 5, wherein said compound which has binding affinity for′both hematopoietic stem cells and said recombinant retrovirus is a component of an extracellular matrix present in a natural hematopoietic environment.
 7. The method according to claim 1, wherein said retroviral vector is derived from a viral MuLV vector.
 8. The method according to claim 7, wherein said retroviral vector comprises as operably linked components with said gene X, a 5′ LTR and a modified 3′ LTR, wherein said 5˜LTR and said 3′ LTR are derived from a ,Jiral MuLV vector, and a 5′ part of a MuLV gag gene.
 9. The method according to claim 8, wherein said MuLV vector is derived from a Mo-MuL¥ vector and wherein said modified 3′ LTR is a hybrid LTR that contains a PyF101 enhancer instead of a Mo-MuLV enhancer.
 10. The method according to claim 9, wherein said retroviral vector comprises vector pLgXL(AMo+PyFI01).
 11. The method according to claim 2, wherein said cells which produce said recombinant retrovirus are mammalian cells which express MuLV gag, pol and env genes.
 12. The method according to claim 11, wherein said env gene is derived from an amphotropic MuLV.
 13. The method according to claim 11, wherein said MuLV gag, poi and env genes are contained in at least two different eukaryotic expression vectors.
 14. The method according to claim 11, wherein each of said expression vectors is associated with a selectable marker gene.
 15. The method according to claim 11, wherein said mammalian cells are GP+envAM 12 cells.
 16. The method according to claim 10 wherein said retrovirus is an amphotropic retrovirus.
 17. The method according to claim 16, wherein cells which produce said recombinant amphotropic retrovirus contain several copies of said retroviral vector.
 18. The method according to claim 2, wherein said cocultivation is in medium which contains serum and at least one hematopoietic growth factor.
 19. The method according to claim 2, wherein following said co-cultivation, non-adherent bone marrow cells are harvested together with adherent bone marrow cells.
 20. A cell which produces a recombinant retrovirus comprising a genome based on a retroviral vector into which is inserted a gene X for introduction into isolated primate bone marrow cells by the method of cocultivating isolated primate bone marrow cells with said recombinant retrovirus.
 21. The cell of claim 20, wherein said retroviral vector is derived from a viral MuLV vector.
 22. The cell of claim 21, wherein said retroviral vector comprises as operably linked components with said gene X, a 5′ LTR and a modified 3′ LTR, wherein said 5′ LTR and said 3′ LTR are derived from a viral MuLV vector, and a 5′ part of a MuLV gag gene.
 23. The cell of claim 22, wherein said viral MuLV vector is derived from a viral Mo-MuLV vector and said modified 3′ LTR is a hybrid LTR that contains a PyF101 enhancer instead of a Mo-MuLV enhancer.
 24. The cell of claim 23, wherein said retroviral vector is pLgXL(&Mo+PyF101), wherein X represents said gene X.
 25. The cell of claim 18, wherein said retrovims is an amphotropic retrovirus.
 26. The cell of claim 25, wherein said cell is a mammalian cell which expresses MuLV gag, pol and env genes.
 27. The cell of claim 26, wherein said env gene is derived from an amphotropic MuLV.
 28. The cell of claim 26, wherein said MuLV gag, poi and env genes are contained in at least two different eukaryotic expression vectors.
 29. The cell of claim 26, wherein each of said expression vectors are associated with a selectable marker gene.
 30. The cell of claim 26, wherein said cells are GP+envAM]2 cells.
 31. The cell of claim 26, wherein said recombinant retrovirus contains several copies of said retroviral vector.
 32. A process for producing genetically modified primate hematopoietic cells with long term repopulating capacity, said method comprising: collecting both adherent and nonadherent genetically modified primate bone marrow cells from a culture in which unmodified primate bone marrow cells were combined with a recombinant retrovirus under conditions whereby retroviral particle concentration in the immediate vicinity of said unmodified bone marrow cells was enhanced so as to increase efficiency of transduction of said bone marrow cells.
 33. Genetically modified primate hematopoietic cells with long term repopulating capacity comprising: a plurality of adherent and nonadherent genetically modified primate bone marrow cells.
 34. Genetically modified primate hematopoietic produced by the method of claim
 1. 35. A method for genetically modifying primate hematopoietic stem cells, said method comprising combining isolated primate hematopoietic stem cells which can undergo replication in culture with a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted, wherein said combining uses means whereby said hematopoietic stem cells and said recombinant retrovims are brought into close physical contact, so that chance of infection of said hematopoietic stem cells by said recombinant retrovirus is significantly enhanced.
 36. The method according to claim 35, wherein said replication is stimulated by adding recombinant hematopoietic growth factors to said culture.
 37. The method according to claim 35, wherein said culture includes a stromal cell support and replication is stimulated by hematopoietic growth factors produced by said stromal cell support.
 38. The method according to claim 35, wherein said combining is cocultivating with a cell line which produces said recombinafit retrovirus and wherein said replication is stimulated by hematopoietic growth factors produced by said cell line.
 39. A cell culture comprising: isolated primate hematopoietic stem cells which can undergo replication and a retrovims producing cell line which produces a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted.
 40. The cell culture of claim 39, further comprising a stromal cell.
 41. The method according to claim 5, wherein said compound which has binding affinity for both hematopoietic stem cells and said recombinant retrovirus is a component present in a natural hematopoietic environment.
 42. The method according to claim 5, wherein said compound which has binding affinity for both hematopoietic stem cells and said recombinant retrovirus is a component of cells derived from a natural hematopoietic environment.
 43. A method for genetically modifying primate hematopoietic stem cells, said method comprising: combining isolated primate hematopoietic stem cells which can undergo replication with a recombinant retrovirus comprising a genome based on a retroviral vector into which a gene X is inserted, wherein said combining uses means whereby said hematopoietic stem cells and said recombinant retrovirus are brought into close physical contact, so that chance of infection of said hematopoietic stem cells by said recombinant retrovirus is significantly enhanced.
 44. A cell culture comprising: isolated primate hematopoietic stem cells which can undergo replication and a stromal cell population, wherein said stromal cell population comprises cultured cells, extracellular matrix molecules and cytokines produced by said cultured cells.
 45. The cell culture according to claim 44, wherein said stromal cell population is derived from human bone marrow mononuclear cells. 